Application Of PI4KIIIA Protein And Related Membrane Protein Complex In Treating Alzheimer's Disease
20240173287 ยท 2024-05-30
Inventors
- Fude HUANG (Shanghai, CN)
- Xiao Zhang (Shanghai, CN)
- Wenan Wang (Shanghai, CN)
- Le Zhang (Shanghai, CN)
- Lixiang Jiang (Shanghai, CN)
- Tong Zhu (Shanghai, CN)
- Haiyan Liu (Shanghai, CN)
- Yudong Zhou (Zheijiang, CN)
- Yang He (Zheijiang, CN)
- Wanguo Wei (Shanghai, CN)
Cpc classification
A61K31/538
HUMAN NECESSITIES
C12Y207/01067
CHEMISTRY; METALLURGY
A61K31/54
HUMAN NECESSITIES
A61K31/4406
HUMAN NECESSITIES
A61K31/343
HUMAN NECESSITIES
A61K31/341
HUMAN NECESSITIES
International classification
A61K31/341
HUMAN NECESSITIES
A61K31/343
HUMAN NECESSITIES
A61K31/4406
HUMAN NECESSITIES
A61K31/538
HUMAN NECESSITIES
A61K31/54
HUMAN NECESSITIES
Abstract
The use of a genetic method to down-regulate RBO/EFR3/EFR3A/EFR3B proteins, TTC7 protein or PI4KIII? enzyme protein which interacts with RBO/EFR3/EFR3A/EFR3B proteins and TTC7 protein, or the use of a drug to inhibit PI4KIII? protein kinase activity reduces the accumulation of A?.sub.42 within neurons and age-dependent synaptic transmission failure and other obstacles in a fruit fly AD model, and obtains an effect of improving the learning and memory abilities of AD model mice. Provided is a method for using an RBO/EFR3/EFR3A/EFR3B inhibitor, a TTC7 inhibitor and a PI4KIII? inhibitor to treat Alzheimer's disease. Also provided is a method for screening a drug treating Alzheimer's disease by whether A? secretion by nerve cells is promoted or not.
Claims
1. A method for preventing or delaying the onset of Alzheimer's disease or any symptoms associated therewith, or ameliorating symptoms or reducing the severity of conditions before the onset of Alzheimer's disease in a subject in need thereof, wherein the method comprises administering to the subject a PI4KIII? inhibitor.
2. The method of claim 1, wherein said PI4KIII? inhibitor is an antibody against PI4KIII?, an inhibitory nucleotide specific to PI4KIII?, or a small molecule compound inhibitor specific to PI4KIII?.
3. The method of claim 2, wherein said inhibitory nucleotide specific to PI4KIII? has the nucleotide sequence as shown in SEQ ID NO: 6.
4. The method of claim 2, wherein said PI4KIII? inhibitor is selected from a small molecule compound inhibitors selected from PAO, a PAO derivative, G1, A1, analogues of G1 or A1.
5. The method of claim 4, wherein said PAO derivative is selected from the group consisting of: ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
6-9. (canceled)
10. The method of claim 1, further comprising administering to the subject at least one of a RBO/EFR3/EFR3A/EFR3B inhibitor and a PI.sub.4P inhibitor.
11. The method of claim 1, further comprising administering to the subject one or more of an antibody against A? and/or a compound capable of removing extraneuronal A? plaques or deposits, wherein the compound capable of removing extraneuronal A? plaques or deposits is selected from the group consisting of marine-derived sulfated oligosaccharide HSH971 and analogues thereof, acamprosate and analogues thereof, and edaravone and analogues thereof.
12. (canceled)
13. The method of claim 1, wherein the PI4KIII? inhibitor inhibits the phosphokinase activity of PI4KIII?.
14. The method of claim 1, wherein the PI4KIII? inhibitor inhibits the interaction between RBO/EFR3/EFR3A/EFR3B protein and PI4KIII? protein, thereby reducing the formation of RBO/EFR3/EFR3A/EFR3B-PI4KIII? protein complexes.
15. The method of claim 1, wherein the PI4KIII? inhibitor inhibits the plasmalemma PI.sub.4P level in the subject.
16. The method of claim 1, wherein the PI4KIII? inhibitor facilitates A?.sub.42 secretion of neurons and reduces A?.sub.42 accumulation in neurons.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0035] In the present invention including the description and claims unless otherwise specified the following terms are used with the following meanings:
[0036] The term rbo/Efr3/Efr3a/Efr3b gene used herein refers to rbo gene originated from Drosophila, Efr3 gene originated from yeast, or Efr3a gene and Efr3b gene originated from mammals; term RBO/EFR3/EFR3A/EFR3B protein used herein refers to proteins encoded by rbo gene originated from Drosophila, Efr3 gene originated from yeast, or Efr3a/Efr3b gene originated from mammals.
[0037] The term PI4KIII?/PI4KA gene used herein refers to PI4KIII? gene or PI4KA gene originated from Drosophila or mammals; term PI4KIII? protein used herein refers to proteins encoded by PI4KIII?/PI4KA gene in Drosophila or mammals.
[0038] The term ttc7 gene used herein refers to ttc7 gene originated from Drosophila and mammals; term TTC7 protein used herein refers to proteins encoded by ttc7 in afore-mentioned Drosophila and mammals.
[0039] The term inhibitor used herein refers to materials capable of lowering, reducing or eliminating the amount, particular function, and particular property of a target object. Said target object can be a protein, polypeptide, nucleic acid and the like, while said inhibitor affects the amount, particular function, and particular property of the target object either directly or indirectly so as to result in the corresponding lowering, reducing or eliminating of the amount, particular function, and particular property of the target object. Said inhibitor can be a protein, polypeptide, nucleic acid, small molecule compound and the like.
[0040] For example, the term PI4KIII? inhibitor used herein refers to materials capable of lowering, reducing or eliminating the expression, transcription, translation of PI4KIII?/PI4KA gene, and/or stability of PI4KIII? protein produced therefrom, binding ability to RBO/EFR3/EFR3A/EFR3B protein and TTC7 protein, and phosphokinase activity thereof, etc., which includes but is not limited to inhibitory nucleotides specific to PI4KIII?/PI4KA, antibodies against PI4KIII? protein, small molecule compound inhibitors capable of inhibiting PI4KIII? kinase activity, and/or materials capable of inhibiting the interaction between PI4KIII? protein and other membrane proteins, and the like.
[0041] Similarly, the term RBO/EFR3/EFR3A/EFR3B inhibitor used herein refers to materials capable of inhibiting, lowering, or eliminating the expression, transcription, translation of rbo/Efr3/Efr3a/Efr3b gene, and/or stability of RBO/EFR3/EFR3A/EFR3B protein produced therefrom, and binding ability to PI4KIII? protein, etc., which includes but is not limited to inhibitory nucleotides specific to rbo/Efr3/Efr3a/Efr3b, antibodies against RBO/EFR3/EFR3A/EFR3B protein, and materials capable of inhibiting formation of complexes of RBO/EFR3/EFR3A/EFR3B protein and PI4KIII? protein, and the like.
[0042] The term TTC7 inhibitor used herein refers to materials capable of lowering, reducing or eliminating the expression, transcription, translation of ttc7 gene, and/or stability of TTC7 protein produced therefrom, and binding ability to RBO/EFR3/EFR3A/EFR3B protein, etc., which includes but is not limited to inhibitory nucleotides specific to ttc7, antibodies against TTC7 protein, and/or materials capable of inhibiting interaction between TTC7 protein and membrane protein RBO/EFR3/EFR3A/EFR3B, and the like.
[0043] Same as above, the term PI.sub.4P inhibitor used herein refers to materials capable of inhibiting, lowering, or eliminating the quantity level of PI.sub.4P on cell membrane, which includes but is not limited to antibodies against PI.sub.4P, and OSH2-PH2X fusion protein or OSH2-2x-PH fusion protein which is capable of specific binding to PI.sub.4P.
[0044] The term antibody used herein refers to any immunoglobulin or complete molecule and fragments thereof which binds to a specific epitope. Said antibody includes but not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or parts of intact antibodies, as long as such fragments or parts retain the antigen binding capacity of the parent antibody. In the invention, for example, antibody against PI4KIII? refers to monoclonal antibodies, polyclonal antibodies, single chain antibodies and immunological activie fragments or parts thereof capable of specific binding to PI4KIII? protein, or functional variants or functional fragments thereof. In the invention, terms such as PI4KIII? antibody, antibody against PI4KIII?, and anti-PI4KIII? antibody are used interchangeably.
[0045] In the invention, functional variant refers to the protein or polypeptide of the invention with one or more amino acid modification in its amino acid sequence. The modification can be a conservative modification (wherein the substituted amino acid has similar structure or chemical property) or a non-conservative modification; similar modification also include addition or deletion of amino acid or both. However, neither the modification of amino acid residue nor the addition or deletion of amino acid would substantially change or damage the biological or immunological activity and function of the original amino acid sequence. In the invention, similarly, functional fragment refers to any part of the protein or polypeptide of the invention, which retains the substantially similar or identical biological or immunological activity and function of the protein or polypeptide of which it is a part (the parent protein or polypeptide).
[0046] The term inhibitory nucleotide used herein refers to nucleotide compound capable of binding to and inhibiting expression of a specific gene. Typical inhibitory nucleotide includes but not limited to antisense oligonucleotides, triple helix DNAs, RNA aptamers, ribozymes, small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA. These nucleotide compounds bind to said specific genes with higher affinity than other nucleotide sequences, so as to inhibit expression of the specific genes.
[0047] The term small molecule compound used herein refers to organic compounds with molecular weight less than 3k dalton which can be either natural or chemically synthesized. Term derivative used herein refers to compounds generated by modifying the parent organic compound through one or more chemical reactions, which have similar structures as the parent organic compound and similar effects in their functions. Term analogue used herein refers to compounds which were not generated by chemically modifying the parent organic compound but are similar to the parent organic compound in structure and have similar effects in their functions.
[0048] The term Alzheimer's disease (AD) used herein refers to an age-related
[0049] neurodegenerative disease characterized in a progressive learning and memory dysfunction. Most AD patients in middle and advanced stages have neural extracellular beta amyloid plaques, intracellular neurofibrillary tangles formed of Tau protein, or loss of synapse and nerve cells. The disease may exist in human or in animals, such as dogs.
[0050] The term A? used herein refers to a series of polypeptides with a length of 38-48 amino acids generated by secretase cleavage of amyloid precursor protein (APP), which include polypeptides A?.sub.38, A?.sub.40, A?.sub.42, A?.sub.44, A?.sub.45 and the like having same amino acid sequences. In the invention, A? can also be generated by cleavage of other protein cleavage enzyme of A? fusion protein expressed by transgenic method or infecting cells with viral vectors through particular expression system, for example, A? through its N-terminus may form fusion proteins with the secretory signal peptide originated from proteins encoded by Drosophila necrotic gene (amino acid sequence: MASKVSILLLLTVHLLAAQTFAQDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA) (SEQ ID NO: 1) or secretory signal peptide originated from rat pre-proenkephalin (amino acid sequence: MAQFLRLCIWLLALGSCLLATVQA) (SEQ ID NO: 2) or the like.
[0051] The term A? secretion used herein refers to a process of discharge of A? generated itracellularly or on cel membranes via cell membrane, which may decrease intracellular A? accumulation. Wherein, A?.sub.42 secretion specifically refers to a process of the discharge of A?.sub.42 generated intracellularly or on cell membranes via cell membrane, which may decrease intracellular A?.sub.42 accumulation.
[0052] The term therapeutic target used herein refers to various materials that can be used to treat a certain disease and the target of the material in animal or human bodies. Treatment effects on said disease are obtainable when said materials act on said target. Said materials can be a variety of materials such as protein, polypeptide, nucleic acid, small molecule compound, said target can be material substances such as a certain gene (including a specific sequence of a gene), a ceratin protein (including a specific site of a protein), a certain protein complex (including specific binding site thereof), or certain charactistics, certain functions, certain interaction relationships with peripheral substances and environment of aforementioned genes and/or proteins, etc, as long as said materials can affect the gene, protein, protein complex, or characteristic, function, interaction relationship thereof so as to treat the disease.
[0053] The terms treat, treating, or treatment used herein refer to reversing, ameliorating or inhibiting the progression of the disease to which the term is applied, or one or more symptoms of the disease. As used herein, depending on the condition of the patient, the term also include prevention of disease, which includes the prevention of disease or the onset of any symptoms associated therewith, and ameliorating symptoms or reducing the severity of any condition before its onset.
[0054] The terms inhibit, weaken, down-regulate, remove and the like all refer to reduction or decreasing in quantity or degree. Such reduction or decreasing is not limited to any extent as long as it exhibits such a trend. For example, the reduction or decreasing can be 100% relative to the original quantity or degree, or can be 50% or even 1% or less.
[0055] The present invention reveals a variety of actions such as down-regulating the expressions of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, or PI4KIII? protein, weakening the interactions between RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII? protein, and inhibiting enzyme activity of PI4KIII? can improve the age-related dysfunctions such as synaptic deficit and loss of nerve cells expressing A?.sub.42, and discloses that these effects are achieved by facilitating A? (particularly A?.sub.42) secretion of neuron cells and reducing A? (particularly A?.sub.42) accumulation on neuron cell membranes or in cells.
[0056] In the invention, for example, the inventor discovered that Efr3a gene knockdown can reduce the atrophy of dendrites and spines of hippocampal neurons in APP/PS1 transgenic mice, while gavage with phenylarsine oxide (PAO), a common inhibitor of PI4KIII? protein, can significantly ameliorate the learning and memory of APP/PSI mice, and can reduce the content of plasmalemma coupled A?.sub.42 (particularly the A?.sub.42 in A?.sub.42 aggregates/oligomers) in brain tissue, although the process may accompanied with increasing of A?.sub.42 content in cerebrospinal fluid. These results demonstrated that dementia symptoms of APP/PSI mice can be ameliorated by facilitating neuronal secretion of A?.sub.42 and reducing accumulation of A?.sub.42 (particularly aggregated A?.sub.42) in neurons or on neuron membranes.
[0057] The inventors discovered that down-regulating the expressions of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, or PI4KIII? protein in cells or neurons, or preventing their formation of protein complexes to decrease the localization of PI4KIII? protein on membranes, or inhibiting phosphokinase activity of PI4KIII? protein, can facilitate of cells and neurons, reducing A?.sub.42 accumulation in neurons, and ameliorating AD-related neorodegeneration and dysfunctions thereof; meanwhile, neither expression levels of A?.sub.42 or APP, nor activities of ?, ?, and ? secretase which cleave APP were significantly affected. In addition, the inventors also discovered that PI.sub.4P, a product of PI4KIII? protein, facilitate aggregation of A?.sub.42 monomers in liposomes, while such facilitation is much stronger than that of the precursor of PI.sub.4P (PI) and its direvative PI.sub.4.5P.
[0058] The inventors believe that A? (including A?.sub.42) are generated from plasmalemmal or intracellular organs; thus generated A? may be secreted from cells through passive release, exocytosis, lysosomal-mediated release, or other undiscovered pathways. Despite of the origin of A? or how it is secreted, plasmalemma is the last pathway through which A? leaves the cell. Due to the hydrophobicity of A?, on plasmalemma, A? is inserted into hydrophobic fatty acid chain region on one hand, and interacts with phosphatidylinositol (particularly phosphorylated phosphatidylinositol PI.sub.4P) and other acidic phospholipid on the other hand, such interaction facilitates the conformation changes of A? from random coil into ?-structure, and further aggregates as A? aggregate to be deposited on membranes or be accumulated in cells by endocytosis. Furthermore, studies have demonstrated that the affinity of soluble A? (including A?.sub.42) aggregates/oligomers to cell or liposome membrane is much higher than that of A? monomer. Therefore, aggregated A? is much easier to accumulate on cell membranes.
[0059] PI.sub.4P is a major component of plasmalemmal phosphorylated phosphatidylinositol, which exhibits a stronger facilitation on the formation of A? aggregates/oligomers than PI and PIP.sub.2. In the invention, the inventors discovered that the facilitation effect of PI.sub.4P on the formation A?.sub.42 aggregates/oligomers in liposomes is clearly dose dependent. Down-regulating the expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, or PI4KIII? protein, preventing their formation of protein complexes, or inhibiting the kinase activity of PI4KIII? protein, can reduce PI.sub.4P production on cell membranes. Therefore, down-regulating expression of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, or PI4KIII? protein, or preventing the formation of membrane attached protein complexes of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII? protein, or inhibiting the corresponding phosphokinase activity of PI4KIII? protein, can substantially decrease the amount of plasmalemma phosphorylated phosphatidylinositol (particularly PI.sub.4P) so as to weaken the interaction between plasmalemmal A? (including A?.sub.42) and phosphorylated phosphatidylinositol, and result in more plasmalemmal A? existing in the random form of A? monomer. As described above, such random form of A? monomer has low affinity to membrane, thus is relatively easily released from membrane and secreted extracellularly. Therefore, above regulation behaviors can effectively reduce intracellular accumulation of A? without affecting the expression level of APP or the activities of ?, ?, and ? secretase, so as to result in an obvious increase of extracellular A? level.
[0060] Previous studies have reported that A?.sub.42 accumulation in Drosophila can activate PI3K and related PI3K/Akt signaling pathway, thus inducing the AD-related synaptic deficits and loss of long-term memory; correspondingly, related research believed that inhibiting PI3K activity can be a method of treating AD. However, in the present invention, the inventors discovered that intracellular A?.sub.42 accumulation is not caused by PI3K/Akt signaling pathway activation of phosphatidylinositol kinases (including PI3K), but is more directly caused by phosphatidylinositol on phosphorylated membranes after plasmalemmal phosphatidylinositol kinase is activated; moreover, the phosphatidylinositol kinase involved in the present invention is mainly PI4KIII? rather than the PI3K in the PI3K/Akt signaling pathway. For example, the inventors discovered that by using phosphatidylinositol kinase inhibitor highly specific to PI4KIII? but not sensitive to PI3K, such as PAO, A?.sub.42 secretion from cells can be effectively facilitated with low concentration.
[0061] It is thus revealed by the inventors that in order to achieve AD treatment, it is possible to adopt a method of facilitating A? secretion, particularly A?.sub.42 secretion, so as to decrease A? (including A?.sub.42) accumulation in neural cells or on cell membranes. But the increased secretion of A? cannot be attributed to the up-regulation of APP or the increased production of A? caused by a change in the activities of ?, ?, and ? secretases.
[0062] It is understandable by one of ordinary skill in the art that there are a variety of routes to facilitate A? secretion of neural cells, including weakening the binding or interaction between A? and plasmalemma saccharides, lipids, and proteins. In the invention, it is preferred to facilitate A? secretion of cells by reducing A? (particularly A?.sub.42) aggregation on cell membranes, as described above.
[0063] Furthermore, the present invention reveals that by regulating the quantities of RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII? protein and their capacity of forming complexes, as well as by regulating phosphokinase activity of PI4KIII? protein, A? aggregates/oligomers formed on cell membranes can be reduced so as to facilitate A? (particularly A?.sub.42) secretion of cells, therefore, these proteins and the relationships thereof can constitute potential theraputic targets for treating AD.
[0064] Accordingly, it is understandable by one of ordinary skill in the art that inhibitors or methods capable of inhibiting, lowering, reducing, or eliminating the expression, transcription, or translation of rbo/Efr3/Efr3a/Efr3b gene, and capable of decreasing the stability of RBO/EFR3/EFR3A/EFR3B protein encoded therefrom, as well as inhibitors or methods capable of inhibiting its formation of protein complexes with phosphatidylinositol kinase PI4KIII? protein and TTC7 protein, can be used to treat AD. Said RBO/EFR3/EFR3A/EFR3B inhibitors include but not limited to inhibitory nucleotides of rbo/Efr3/Efr3a/Efr3b gene (including antisense RNA, siRNA, miRNA or the like), antibodies against RBO/EFR3/EFR3A/EFR3B protein, and the like.
[0065] It is understood by the skilled in the art that inhibitory nucleotides of rbo/Efr3/Efr3a/Efr3b gene are well-known in the art (e.g., see, www.genecards.org, and available products: ORIGENE, Cat. #SR308056 and Cat. #TR303768). Similarly, antibodies against RBO/EFR3/EFR3A/EFR3B protein are well-known in the art (e.g., see, www.genecards.org, and available products: Novus, Cat. #NBP1-81539; Thermo Fisher Scientific, Cat. #PA5-24904).
[0066] Moreover, as described above, regulating the cellular expression, transcription, or translation of PI4KIII?/PI4KA gene, regulating the stability of PI4KIII? protein encoded from PI4KIII?/PI4KA gene, regulating the capacity of PI4KIII? protein forming complexes with membrane protein RBO/EFR3/EFR3A/EFR3B and TTC7 protein, and regulating phosphokinase activity of PI4KIII? protein, can be regarded as methods of treating AD. Therefore, it is understandable by one of ordinary skill in the art that inhibitors or methods capable of inhibiting, lowering, reducing, or eliminating the expression, transcription, or translation of PI4KIII?/PI4KA gene, or capable of decreasing the stability of PI4KIII? protein encoded therefrom, inhibitory nucleotides includes but not limited to that specific to PI4KIII?/PI4KA gene, antibodies against PI4KIII? protein, and small molecule compound inhibitors capable of inhibiting protein complex formation of PI4KIII? protein with membrane proteins and capable of inhibiting kiniase activities, can be used to treat AD. Preferably, the inhibitor is a small molecule compound, for example, PAO (Phenylarsine Oxide), PAO derivatives, A1, G1, or analogues of A1 and G1. More preferably, the inhibitor is PAO or PAO derivatives.
[0067] It is understood by the skilled in the art that PAO is a small molecule compound having a basic structure comprising oxoarsine group and phenyl group, which exhibits a strong inhibitory effect on the phosphokinase activity of PI4KIII? protein. Chemical structure of PAO is:
##STR00001##
[0068] Synthesis method of PAO is well-known to the skilled in the art. According to the present invention, compounds that can be used in the treatment of AD further include derivatives of PAO, as long as the compound exhibit inhibitory effect on the phosphokinase activity of PI4KIII? protein. It is understood by the skilled in the art that synthesis methods of such derivatives are well-known in the art.
[0069] Similarly, it is understood by the skilled in the art that A1 and G1 both are small molecule compound inhibitors of PI4KIII? protein and having similar structures. The chemical structure of A1 is:
##STR00002## [0070] 5-(2-amino-1-(4-(4-morpholinyl)phenyl)-1H-benzimidazol-6-yl)-N-(2-fluorophenyl)-2-methoxy-3-pyridinesulfonamide.
[0071] Chemical structure of G1 is:
##STR00003## [0072] (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide.
[0073] Synthesis methods of A1 and G1 are well-known in the art (e.g., see, Bojjireddy, N., et al. (2014), JBC 289: 6120-6132; Leivers, A. L., et al. (2014), JMC 57: 2091-2106). According to the present invention, structural analogues of A1 and G1 can be used to treat AD as long as they exhibit inhibitory effect on the phosphokinase activity of PI4KIII? protein. It is understood by the skilled in the art that synthesis methods of such structural analogues are well-known in the art.
[0074] In addition, the formation of membrane complexes of PI4KIII? protein and RBO/EFR3/EFR3A/EFR3B protein requires the assistance of scaffold protein TTC7. Therefore, according to the invention, it is understandable by one of ordinary skill in the art that inhibitors or methods capable of inhibiting, lowering, reducing, or eliminating the expression, transcription, or translation of ttc7 gene, and capable of decreasing the stability of TTC7 protein encoded therefrom, as well as inhibitors or methods capable of inhibiting its formation of protein complexes with RBO/EFR3/EFR3A/EFR3B protein and PI4KIII? protein, can be used to treat AD. Said TTC7 inhibitors include but not limited to inhibitory nucleotides of ttc7 gene (including antisense RNA, siRNA, miRNA or the like), antibodies against TTC7 protein, and the like.
[0075] Moreover, according to the invention, down-regulating the expression of RBO/EFR3/EFR3A/EFR3B protein and PI4KIII? protein, or preventing their formation of protein complexes to decrease the distribution of PI4KIII? protein on membranes, or inhibiting the phosphokinase activity of PI4KIII? protein, essentially leads to reduction of plasmalemmal PI.sub.4P and further facilitates A? secretion of cells. Therefore, it is understandable by one of ordinary skill in the art that any inhibitor or method capable of decreasing the quantity or level of plasmalemmal PI.sub.4P, and further decreasing the formation of A? aggregates/oligomers on cell membranes, can achieve the effect of treating AD as described above.
[0076] It is understandable by the skilled in the art that aforementioned PI.sub.4P inhibitors can be antibodies or other molecules that specifically binds to PI.sub.4P. Currently, synthesis methods of antibodies against PI.sub.4P are well-known in the art (see, Brown BK and Karasavass N, et al., 2007, J virol; Wassef N M and Roerdink F, et al. 1984, Mol Immuol). For example, it can be human-derived broad-neutralizing antibody 4E10, and other antibodies against PI.sub.4P. Currently, synthesis methods of molecules specifically bind to PI.sub.4P are well-known in the art (see, Balla A and Kim Y J, et al., 2008, Mol Biol Cell; Zubenko G S and Stiffler et al., 1999, Biol Psychiary). For example, it can be an OSH2-PH2X fusion protein, or an OSH2-2x-PH fusion protein.
[0077] Aforementioned materials provided by the present invention that can be used to treat AD or having treatment potential to AD (hereinafter collectively referred to as the materials of invention), including but not limited to antibodies against RBO/EFR3/EFR3A/EFR3B, antibodies against PI4KIII?, antibodies against TTC7, antibodies against PI.sub.4P, inhibitory polypeptides specific to rbo/Efr3/Efr3a/Efr3b gene, inhibitory polypeptides specific to PI4KIII?/PI4KA gene, and small molecule compounds inhibiting phosphokinase activity of PI4KIII? protein, etc., can be isolated, purified, synthesized and/or recombined.
[0078] Moreover, the materials of invention can also be formulated into a composition, such as a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising any of aforementioned antibodies, inhibitory polypeptides, and/or small molecule compounds, and a pharmaceutically acceptable carrier.
[0079] The pharmaceutical composition of the invention containing any of the materials of invention can comprise more than one material of invention, e.g., antibody and small molecule compound, inhibitory polypeptide and antibody, or two or more antibodies or small molecule compounds. Alternatively, the pharmaceutical composition can also comprise a material of invention in combination with another pharmaceutically active agent or drug. For example, it can comprise antibody drug against A?, such as Bapineuzumab, or compound which binds to neural extracellular A? or ? amyloid plaque in brain to block A? aggregation or to facilitate disaggregation of A? aggregates, such as marine-derived sulfated oligosaccharide HSH971 and analogues thereof, acamprosate (tramiprosate) and analogues thereof, and Edaravone and analogues thereof. In this way, the pharmaceutical composition facilitates A? secretion from neurons on the one hand, and facilitates the removal of A? outside neurons on the other hand, thereby achieving a better therapeutic effect on the treatment of AD.
[0080] In another aspect, the present invention provides a method for screening new medicines or therapeutic targets for the treatment of AD. The method is designed based on aforementioned discoveries of the invention, i.e., intracellular A?.sub.42 accumulation can be reduced by facilitating A?.sub.42 secretion so as to ameliorate and prevent AD-associated neurodegeneration and dysfunction. Therefore, the criteria for screening medicines or therapeutic targets is the facilitation effect on A? secretion (particularly A?.sub.42 secretion) after administration of the medicine or regulation of the therapeutic target, while increased A? secretion cannot be attributed to the up-regulation of APP or the increased production of A? caused by change in activities of ?, ?, and ? secretase.
[0081] According to the present invention, regulation of the therapeutic target refers to using relevant materials to act on the therapeutic target either directly or indirectly so as to change the function, property, or relationship with peripheral environment of the therapeutic target, thus causing or inducing the facilitation of A? secretion (particularly A?.sub.42 secretion) of cells.
[0082] It is understood by one of ordinary skill in the art that in order to select effective drug for treating Alzheimer's disease, cell lines for screening test can be eukaryotic cell lines from mammals, insects or the like, e.g., HEK293, COS7, N2a, SH-SY5Y, S2, sf9 and the like. The method includes testing whether a candidate drug may reduce A? accumulation, particularly A?.sub.42 accumulation, on cell membrane or in cells, so as to select effective drugs for treating Alzheimer's disease. Preferably, the test of whether A? secretion is increased can be performed in cell lines over-expressing APP (e.g., HEK293, COS7, N2a, SH-SY5Y cell lines stably transfected with human-derived APP) or with Drosophila model, preferably tissues of third instar larva of Drosophila. Whether A? secretion is increased can be detected with methods of immunoassay, including enzyme-linked immunosorbent assay (ELISA) or electro-chemiluminescence assay (ECLIA).
[0083] Preferably, the method for screening medicines or therapeutic targets for the treatment of AD can include: observing the effect of invention of candidate medicine or target regulation on enzyme activity of PI4KIII?, if the invention of candidate medicine or target regulation negatively affects the function of PI4KIII? kinase in the detection system, i.e., characterized in reduction of PI4KIII? kinase activity or plasmalemmal PI.sub.4P level, then it indicates that the candidate medicine, agent or target is a potential medicine or therapeutic target for treating AD. After such screening, it is further detected that whether intracellular A? (particularly A?.sub.42) accumulation is reduced and whether extracellular A? secretion is facilitated. With these methods, screening efficiency of candidates can be greatly improved.
[0084] According to one specific embodiment of the present invention, the method for screening medicines or therapeutic targets for the treatment of AD can include: determining based on directly analyzing whether invention of candidate medicine or regulation of therapeutic target re-distributes plasmalemmal PI4KIII? protein to endochylema so as to reduce the quantity of PI4KIII? protein on membrane and induces a reduction of the aggregation/oligomerization of plasmalemma A? monomer as well as increasing extracellular secretion. Preferably, fluorescently labeled PI4KIII? can be selected for observation, such as PI4KIII? labeled with fluorescent protein (GFP-PI4KIII?), and observing whether the fluorescently labeled PI4KIII? transfers from plasmalemma to endochylema.
[0085] According to one specific embodiment of the present invention, the method for screening medicines or therapeutic targets for the treatment of AD can also be conducted with methods such as co-immunoprecipitation assay in which interactions between proteins are analyzed. If invention of candidate medicine or regulation of therapeutic target reduces interactions between RBO/EFR3/EFR3A/EFR3B protein, TTC7 protein, and PI4KIII? protein, it indicates that the medicine or therapeutic target is capable of weakening the capacity of PI4KIII? protein in forming membrane protein complexes so as to reduce aggregation of plasmalemma A? monomer as well as increasing extracellular secretion.
[0086] According to one specific embodiment of the present invention, the method for screening medicines or therapeutic targets for the treatment of AD can also be performed by directly analyzing whether invention of candidate medicine or regulation of therapeutic target reduces plasmalemmal PI.sub.4P level. Preferably, fluorescence microscope, confocal microscope, or two-photon microscope can be utilized to observe whether fluorescently labeled molecule that specifically binds to PI.sub.4P, such as OSH2-PH2X or OSH2-2x-PH fusion protein labeled with fluorescent protein, is decreased in plasmalemma quantity or transferred from plasmalemma to endochylema.
[0087] The present invention will be further illustrated in detail below. However, ways to carry out the present invention are not limited to the following examples.
[0088] Hereinafter, data are obtained mainly through animal and cell culture experiments, and are analyzed with SPSS software. Unless otherwise specified, data are represented by mean?sem. P<0.05 indicates the difference is statistically significant. All data shown here and below are represented by mean?sem. *, **, and *** each represents that p<0.05, 0.01, and 0.001, respectively.
EXAMPLE 1
Drosophila Strains and Genetics Methods
[0089] Standard culture medium, alternating cycle of 12-hour light and 12-hour dark, culturing under constant temperature of 25? C.
[0090] The following transgenic Drosophila strains were utilized in the invention: rbo.sup.S358A, [UAS]A?.sub.arc, [UAS]A?.sub.42, [UAS]dtau, [UAS]mCD8-gfp (provided by Dr. Z. Wang), [UAS]shibire.sup.ts1 (provided by Dr. A Guo), [Gal4]A307 (provided by Dr. O'Kane). Wherein, rbo.sup.S358A gene is a transgene constructed from wild type genome DNA comprising rbo gene via site-directed mutation, whose expression is driven by the pre-driver of the rbo gene itself. [Gal4]A307 expresses transcription factor Gal4, which drives [UAS]A?.sub.arc, [UAS]A?.sub.42, [UAS]dtau, [UAS]mCD8-gfp, [UAS]shibire.sup.ts1 transgenes to express A?.sub.arc, A?.sub.42, dTau, mCD8-GFP, or temperature sensitive mutation Dynamin in neurons of Giant Fiber pathway and in a small amount of other neurons. Drosophila mutants utilized in the invention include: rbo.sup.ts1 (temperature sensitive missense mutation), rbo.sup.2 (knockdown mutation), itpr.sup.sv135 (nonsense mutation, Bloomington Stock #30740), PI4KIII?.sup.def (mutation with deletion of PI4KIII? gene and peripheral DNA, Bloomington Stock #9518), PI4KIII?.sup.GS27, and PI4KIII?.sup.GJ86 (both are nonsense mutations). P{lacW}1(2)k14710k.sup.15603 transposon is inserted into the first exon of l(2) k14710 gene in order to prevent transcription of l(2) k14710 (Bloomington Stock #11134); P{EPgy2}bin3.sup.EY09582 (Bloomington Stock #20043). In order to purify the genetic background, all transgenes and mutant flies were backcrossed with a wild isogenic strain (isogenic w.sup.1118, Bloomington stock #5905) for more than 5 generations before use.
[0091] Prior research of the inventors discovered that flies (Drosophila) expressing wild type or the arctic mutant A?.sub.42 (A?42 or A?.sub.arc flies or Drosophila) in neurons of the GF pathway exhibit intraneuronal A?.sub.42 accumulation, age dependent synaptic transmission failure, and premature death. Such flies also exhibit an age-dependent decline of climbing ability. In order to study the role of rbo gene in the neural deficits caused by intraneuronal A?.sub.42 accumulation, two mutations of rbo gene, missense mutation (rbo.sup.ts1) and knockdown mutation (rbo.sup.2), were introduced into A?.sub.arc flies, and the effects on synaptic transmission, climbing ability, and age were respectively tested. Four groups of flies were constructed: control flies (control, ctrl), rbo.sup.ts1/+ or rbo.sup.2/+ heterozygotes (rbo), A?.sub.arc flies (A?.sub.arc), and A?.sub.arc flies with rbo.sup.ts1/+ or rbo.sup.2/+ heterozygous mutation (A?.sub.arc-rbo). Each group contains 1-2 strains, wherein, ctrl denotes wild type control flies having [Gal4]A307 transgene; rbo.sup.ts1/+andrbo.sup.2/+ denotes rbo.sup.ts1/+ and rbo.sup.2/+ heterozygous flies having one copy of [Gal4]A307 transgene; A?.sub.arc denotes [Gal4]A307/[UAS]A?.sub.arc double transgenic flies; A?.sub.arc-rbo.sup.ts1/+ and A?.sub.arc-rbo.sup.2/+ each denotes [Gal4]A307-rbo.sup.ts1/[UAS]A?.sub.arc and [Gal4]A307-rbo.sup.2/[UAS]A?.sub.arc, respectively. The first two groups of flies did not express A?.sub.arc and were classified as non-A? flies, whereas the latter two groups of flies express A?.sub.arc and were classified as A? flies.
EXAMPLE 2
rbo Gene Mutation Specifically Suppresses Neural Deficits in A?42-Expressing Drosophila, Tested by Synaptic Transmission Examination
[0092] Examination method of synaptic transmission: Recording of excitatory junction potentials (EJPs) in Giant Fiber (GF) system intracellularly. Adult female fly of a certain day-age was mounted ventral side down on a glass slides with low-melting wax tackiwax (Bockel Scientific) under a dissection microscope. Recording system includes one reference electrode in abdomen, two stimulation electrodes inserted into two eyes, and one recording electrode inserted into dorsal longitudinal muscle cell. Both eyes were subjected to electric stimulation (100Hz, 50 pulses). Stimulation intensity is 5-20 volts with a duration of 0.2 ms, approximately 150% of the threshold stimulation intensity. Electric signals were recorded and amplified by Axonal clamp 900A (Molecular Devices), and were digitized at a frequency of 10 kHz by Digidata 1440A (Molecular Devices). Data were recorded and analyzed by pClamp software (version 10.0; Molecular Devices). All electrodes are glass electrode filled with 3M KCl solution. Environment temperature was 25? C. during recording.
[0093]
[0094] According to the above examination method of synaptic transmission, intracellular recording of EJPs in the dorsal longitudinal muscle fibers under high-frequency electric stimulation (100Hz, 50 pulses) was performed on the 3-7.sup.th, 15-17.sup.th, 25-27.sup.th, and 31-35.sup.th days after eclosion. The success rate of EJPs elicited by high-frequency electric stimulation in the first group was not significantly differentiable from that in the other three groups on the 3-7.sup.th and 15-17.sup.th days (
[0095] These results illustrates that rbo gene mutation ameliorates the age-dependent synaptic transmission failure caused by intraneuronal A?.sub.arc accumulation. It is unlikely that the difference in genetic background has contributed to the amelioration, since the genetic background of the transgenic flies and rbo mutants used for creating the four groups of flies were backcrossed with a wild isogenic strain (isogenic w.sup.1118) for more than 5 generations before use, thus the genetic background is essentially purified. Since total knockdown of rbo gene causes embryonic lethality, its effect on A?.sub.arc induced synaptic transmission failure could not be examined.
EXAMPLE 3
rbo Gene Mutation Specifically Suppresses Neural Deficits in A?42-Expressing Flies, Tested by Tube-Climbing Ability Assay
[0096] Tube climbing test: Climbing ability was examined by measuring the average climbing height of 10 flies at the seventh second from the bottom of vertically placed testing tubes. A fruit fly climbing ability testing apparatus with high reproducibility was developed. The apparatus includes: 1) a rectangular metal frame (32 cm width, 21 cm height) within which 10 transparent plastic tubes are mounted vertically; 2) an electric motor for driving the vertical movement of the metal frame; 3) a stepping actuator for controlling the electric motor at the working cycle of rapidly moving the metal frame up and down for four times for a predetermined height at a 1 minute interval; 4) a video camera for videotaping the climbing process; 5) an analyzing software for analyzing the climbing position of a fly at a certain time of the video. In experiments, 10 flies of a specific genotype were transferred into each transparent plastic tube. The tubes were evenly distributed and mounted in the metal box. The metal box can slide vertically along two metal rods which were mounted vertically on the base. In the climbing test, the stepping actuator controls the electric motor to lift up the metal box for 5.8 cm and then releases, such that the metal box drops to the original position by gravity. Upon the metal box stopped moving, files dropped down to the bottom of the tubes. After the metal box was moved up and down for 4 times in 3 seconds, all files were at the bottom of the tubes. Then the flies were allowed to climb up along the wall of the tubes. The whole processes were videotaped for subsequent analysis. The inventors developed a computer program for measuring the height of a fly at any given time after tube climbing was started.
[0097] In
[0098] Climbing ability was examined on the 3.sup.rd, 16.sup.th, 26.sup.th, and 31.sup.st day after eclosion. On the 3.sup.rd and 16.sup.th day, the climbing abilities of flies in four groups were similar (
EXAMPLE 4
rbo Gene Mutation Specifically Suppresses Neural Deficits in A?42-Expressing Flies, Tested by Longevity Assay
[0099] Longevity (lifespan) assay: 100 or 200 flies of each genotype were equally separated into 5 or 10 tubes containing standard fly food and dry yeast, and cultured at 25? C. Flies were transferred to tubes with fresh food and dry yeast every 3 days, and dead flies were counted at each transfer. Survival rates were analyzed with the SPSS 11 Kaplan-Meier software.
[0100] In
[0101]
[0102] In
[0103] In
[0104] Longevity assay showed that lifespan of A?.sub.arc-rbo flies were longer than that of A?.sub.arc flies, although shorter than that of control and rbo flies (
TABLE-US-00001 TABLE 1 Mean lifespan of flies and comparison thereof Mean p value p value lifespan (compared to (compared Flies (days) control group) to A?.sub.arc group) ctrl 47.5 n.a. <0.001 rbo.sup.ts1/+ 46.2 0.201 <0.001 rbo.sup.2/+ 54.3 <0.001 <0.001 A?.sub.arc 27.0 <0.001 n.a. A?.sub.arc-rbo.sup.ts1/+ 39.9 <0.001 <0.001 A?.sub.arc-rbo.sup.2/+ 36.8 <0.001 <0.001
[0105]
[0106] The effect of rbo gene mutation against the A?.sub.42 toxicity could not be ascribed to a general effect against intraneuronal accumulation of toxic proteins because rbo gene mutation could not ameliorate the lifespan shortening of flies over-expressing tau protein (
[0107] With examples 1-4, the results showed that rbo gene mutation or insufficiency can specifically suppresses neural deficits in wild type and mutant A?.sub.42-expressing flies.
EXAMPLE 5
Deficiency or Inhibition of PI4KIII? Enzyme Which Interacts With RBO Protein Ameliorates the Neural Deficits in A?.SUB.arc .Flies, Tested by Immunoprecipitation Assay
[0108] Immunoprecipitation and immunoblot: A total of 300 fly heads were collected and homogenized by milling in 500 ?L pre-cooled Tris buffer. The formulation of Tris buffer contains: 50 mM Tris, 50 mM KCl, 1 mM EDTA, 1% cocktail protease inhibitor(Calbiochem), and pH adjusted to 7.4. Tissue homogenate was centrifuged at 10000 g for 10 min. Supernatant was collected and subjected to immunoprecipitation or immunoblot test with about 1 ?g of a mouse monoclonal antibody against RBO or a rabbit polyclonal antibody against Drosophila PI4KIII?. Two antibodies were generated in collaboration with Abmart (Shanghai) or with Abgent (China), respectively. RBO antibody was generated using the 251.sup.th-500.sup.th amino acids of Drosophila subtype C RBO protein; PI4KIII? antibody was generated using the peptide NH2-KRSNRSKRLQYQKDSYC-CONH2 (SEQ ID NO: 3). In immunoblot tests, antibodies against Drosophila RBO and PI4KIII? were diluted by 1:2000. Head tissue homogenates of wild type and corresponding homozygous mutants were respectively used to detect antibodies against Drosophila RBO and PI4KIII?.
[0109] In
[0110] Although RBO might be a putative diacylglycerol (DAG) lipase, and the activity of DAG lipase was reported to be increased in the hippocampus of AD patients and animal models, RBO protein might not regulate A?.sub.arc toxicity by acting as a DAG lipase because introduction of a transgene rbo.sup.S58A into A?.sub.arc flies could not change the premature death (
[0111]
[0112]
[0113]
[0114]
[0115] The RBO homologs in yeast and mouse recruit PI4KIII? and form a complex with it on cell membrane. Consistent with this, RBO protein specifically coimmunoprecipitated with Drosophila PI4KIII? (
[0116] To test whether PI4KIII? plays a similar role as RBO protein in neural deficits of A?.sub.arc flies, a chromosomal deficiency (deletion of a PI4KIII?-containing DNA segment of a chromosome, pi4k.sup.def/+) and a nonsense mutation of PI4KIII? (PI4KIII?.sup.GS27/+) was separately introduced to A?.sub.arc-expressing flies.
[0117]
[0118] In fact, experimental results demonstrated that such PI4KIII? mutations suppressed the A?.sub.arc induced defects in synaptic transmission, motor function, and lifespan (
[0119] In
[0120] However, the suppression of the neural deficits by down-regulating RBO/PI4KIII? could not attribute to a toxicity effect caused by an attenuation of calcium release mediated by phospholipase C, PI4,5P and the receptor of inositol triphosphate (IP3R) because introducing a nonsense mutation of the gene encoding IP3R into A?.sub.arc-expression flies could not attenuate the synaptic failure or the premature death (
EXAMPLE 6
Down-Regulation of RBO/PI4KIII? Decreases Intracellular A?.SUB.42 .Accumulation, Detected by Staining and Imaging
[0121] Staining and imaging: Central nervous system of Drosophila was stained as followed. The whole central nervous system (CNS) of flies, including the brain and ventral ganglion, was dissected out in cold PBS and fixed with 4% PFA in PBS for about 45 min. Preparations were washed with PBS for 30 min, treated with formic acid (70% in water) for 45 min to reexpose the antigenic determinant, washed repetitively with 5% BSA in PBS solution supplemented with 0.25% Triton, incubated with primary antibody (6E10, 1:100 dilution) at 4? C. for 10-12 h, washed with PBS again, and finally incubated with cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:200 dilution) at room temperature for 2 h. Images were taken under Nikon A1R-A1 confocal microscope; the genotypes of fly CNS were blind to the imaging personnel.
[0122]
[0123] Previously, neuronal damage induced by A?.sub.arc-expression in GF pathway was attributed to intracellular accumulation A? protein. Here, we further confirmed the intraneuronal accumulation of A? by introducing uas-mCD8-gfp transgene into A?.sub.arc flies. uas-mCD8-GFP expresses mCD8-GFP fluorescent protein, which targets the plasma membrane and was driven by the same driver as that of A?.sub.arc, so that the A?.sub.arc-expressing neurons could be labeled with GFP. Confocal imaging revealed that A? immunostaining signal colocalized with GFP signal (
[0124] To analyze whether RBO/PI4KIII? insufficiency affects intracellular A? accumulation, A? immunostaining was performed in A?.sub.arc, A?.sub.arc-rbo, and A?.sub.arc-PI4KIII? flies. It is found that immunostaining signal of A?.sub.arc-rbo and A?.sub.arc-PI4KIII? flies significantly decreased as compared to that of A?.sub.arc flies (
EXAMPLE 7
Down-Regulation of RBO/PI4KIII? Decreases Intracellular A?.SUB.42 .Accumulation, Detected by ELISA Quantitative Analysis
[0125] ELISA method analysis: ELISA was performed using A?.sub.42 Human ELISA Kit (Invitrogen) according to the manufacturer's specifications. To analyze A?.sub.42 level in CNS, intact brains of 25 flies per strain were dissected out in cold PBS and placed immediately into cold ELISA sample dilution buffer supplemented with cocktail protease inhibitor (Calbiochem). Brains were homogenized thoroughly, incubated at room temperature for 4 h, and stored under ?20? C.
[0126] Similar as in Example 6, ELISA quantitative analysis shows that the amount of A?.sub.42 was significantly decreased in A?.sub.arc-rbo flies, A?.sub.arc-PI4KIII? flies, and A?.sub.arc flies after PAO treatment (
[0127]
[0128] With Examples 6-7, it is demonstrated that decreasing of intraneuronal A? accumulation induced by RBO/PI4KIII? down-regulation is unlikely due to reduction of intake of extracellular A?.sub.42. The reasons are: 1) intake of extracellular A?.sub.42 in N2a cells having rbo homolog gene knockdown does not reduce significantly (
EXAMPLE 8
Solution Preparation and Toxicity Examination of PI4KIII? Inhibitors PAO, A1 and G1
[0129] HEK293 cells, Drosophila larva, and adult flies were treated with PAO or A1, stock solutions of 10 mM and 0.9 mM A1 was made separately by dissolving PAO powder (Sigma-Aldrich, CAS NO. 637-03-6) and A1 powder in DMSO. Then gradiently diluted with distilled water to the desired concentrations; the final concentration of DMSO was adjusted to identical level to ensure the experimental results were not influenced by DMSO variation.
[0130] To test the toxicity of PAO on living flies, we cultured wild type flies with fly food containing 50, 100, 200, 300, 400, and 600 ?M PAO, started from embryonic stage. It is found that PAO treatment at 200 ?M or less neither changed the eclosion rate nor altered the climbing ability after eclosion. Thus 25, 50, 100, and 150 ?M PAO were chosen for culturing A?.sub.arc and control flies.
[0131] To test the toxicity of PAO on dissected Drosophila third instar larvae, Schneider's culture medium containing 50, 100, 200, 300, 400, and 500 nM PAO were used to incubate dissected Drosophila third instar larvae at 25? C. overnight. It is found that salivary gland cells and CNS neurons in the larvae treated with PAO at 300 nM or more turned white, reflecting damage, whereas no such effect with PAO at 150 nM or less. Thus 50, 100, and 150 nM PAO were chosen for culturing.
[0132] To test the toxicity of PAO and A1 on HEK293T cells, DMEM culture medium containing 50, 100, 200, 300, 400, and 600 nM PAO or A1 were used to incubate HEK239 cells for 12 h. According to MTT tests, it is found that PAO or A1 at 250 nM or more killed most of the cells, whereas no such effect at 150 nM or less. Thus 25, 50, 100, and 150 nM PAO or A1 were chosen for culturing.
[0133] To test the toxicity of PAO oral gavage on mice, PAO powder was dissolved in DMSO to prepare a stock solution of 30 mg/mL. Then gradiently diluted with distilled water to the desired concentrations; the final concentration of DMSO was adjusted to identical level to ensure the experimental results were not influenced by DMSO variation. C57BL/6 mice of 3 month-age were subjected to gavage at doses of 18, 10 and 6 mg/kg body weight, two mice for each dose level. All mice were found dead on the second day. Then mice were subjected to gavage at doses of 4.5 and 2.0 mg/kg body weight, five mice for each dose level. With respect to 4.5 mg/kg body weight dose, gavage once a day for five consecutive days, four mice out of five survived. With respect to 2.0 mg/kg body weight dose, gavage once a day every Monday through Friday, no gavage on weekends. After two consecutive weeks, all five mice survived. Therefore, with respect to C57/B6 mice, median lethal dose of PAO gavage is 2-6 mg/kg body weight, approximately 4 mg/kg body weight. Thus 0.1, 0.3 and 1.0 mg/kg body weight doses were chosen for PAO gavage in APP/PS1 mice and control mice, gavage once a day every Monday through Friday, no gavage on weekends, for six consecutive wecks.
EXAMPLE 9
Down-Regulation of RBO/PI4KIII? Facilitates A?.SUB.42 .Secretion, Detected by Culturing of A?-Expressing Larvae Tissue
[0134] Culturing of A?-expressing larvae tissue: Third instar larvae were washed with water and sterilized with 70% alcohol for 2 min, and were dissected along the dorsal middle line in Schneider's (Sigma) culture medium. The tracheal, gut, and fat body of larvae were removed with caution. The dissected larvae were washed with Schneider's culture medium and transferred into 2 mL centrifuge tube containing 150 ?L Schneider's culture medium supplemented with gentamycin (20 mg/mL). Each tube contained 5 dissected larvae. The centrifuge tubes were placed in a humid and dark environment at 25? C. for 8 h. Then 100 ?L was taken from each tube and was used for ELISA quantification of A?.sub.42. ELISA was performed using A?.sub.42 Human ELISA Kit (Invitrogen).
[0135] In
[0136] To investigate the mechanism of PAO treatment and RBO/PI4KIII? insufficiency decreasing intraneuraonal A? accumulation, A?.sub.42 secretion while incubation of dissected sample of A?.sub.arc-expressing third instar larvae in Schneider's culture medium was detected. ELISA assay shows that PAO treatment facilitates A?.sub.42 secretion and exhibits a tendency of drug dose dependency (
EXAMPLE 10
Down-Regulation of RBO/PI4KIII? Facilitates A?.SUB.42 .Secretion, Detected by Culturing of Human-Derived APP-Expressing HEK293T Cells
[0137] Culturing of human-derived APP-expressing HEK293T cells: HEK293T cells stably transfected with human APP (here named as HEK293T-APP cells) were cultured in DMEM (Hyclone) supplemented with 10% FBS (Gibco), penicillin, streptomycin, and G418 (100 ?g/mL). Recombinant plasmid pSUPER.basic-expressing shRNA of target genes were transiently transfected into HEK293T using Lipofectamine? 2000 (Invitrogen). Cells were incubated for two days after transfection, followed with subsequent experiments. For ELISA quantification of A?.sub.42 concentration in culture medium, freshly-changed culture medium was examined after culturing cells for 12 h.
[0138] Assay of activities of ?, ?, and ? secretase of APP and APP expression level in HEK293T-APP cells: HEK293T-APP cells were incubated in 12-well plate, culture fluids contained PAO at concentrations of 0, 25, 50, 100, or 150 nM. After 6-8 hours incubation, same amounts of cells were collected separately. To analyze secretase activities, cells were lysed separately with 500 ?L of TBS buffer, centrifuged for 15 min, supernatants were collected and precipitates were resuspended with 500 ?L of TBS buffer. To analyze the activities of ? and ? secretases, 100 ?L supernatant was mixed with 2? reaction solution (50 mM Tris-HCl, pH 6.8, 4 mM EDTA, 0.5% CHAPSO (w/v)) containing 10 ?M of specific fluorogenic substrates of ? or ? secretase (Calbiochem, Cat. No. 565767/565758); to analyze activity of ? secretase, 100 ?L resuspended solution was mixed with 2? reaction solution (50 mM Tris-HCl, pH 6.8, 4 mM EDTA, 0.5% CHAPSO (w/v)) containing 10 ?M of specific fluorogenic substrates of ? secretase (Calbiochem, Cat. No. 565764). After reacting at 37? C. for 30 min, analyze with microplate reader (excitation/emission: 365/490 nm for ?/? enzyme activity, 365/440 for ? enzyme activity).
[0139] When analyzing the expression level of APP, Western Blotting was performed using anti-APP/A? antibody (6E10) on collected cells after lysed with TBS buffer containing protease inhibitor (1% cocktail, invitrogen).
[0140]
[0141] To detect whether such facilitation affect the secretion of A?.sub.42 derived from cleavage of ? amyloid precursor protein (APP), A?.sub.42 secretion of HEK293T-APP cells was tested.
[0142] PAO and PAO derivatives were analyzed according to A?.sub.42 secretion detected in HEK293T-APP cells, and the following results were obtained as listed in Table 2.
TABLE-US-00002 TABLE 2 PAO, PAO derivatives, and activities thereof No. Chemical structure Name Mw Activity 1
[0143] In addition, PAO facilitates A?.sub.42 secretion in HEK293T cells stably transfected with human-derived APP without affecting the activities of ?, ?, and ? secretase which cleaves APP (
[0144] Binding simulations of PAO and derivatives thereof with PI4KIII?: At present, structure of PI4KIII? is yet to be measured and reported. The inventors adopt two commonly used software, SWISS-MODEL and MODELLER, with reference to the reported 4DOL structure of PI4KIII?, 4YKN structure of PI3K? and 1e8x structure of PI3K? as templates to construct 6 structural models of PI4KIII? . It is found that the structures of 6 models are highly consistent with each other; root mean square deviation between the structures is less than 3 ?. Interaction parameters between PAO and PI4KIII? was simulated and calculated with this model. PAO can bind to enzyme active center of PI4KIII? from human and other mammals, while forming two strong hydrogen bonds (bond length ?2.03 ?) with the characteristic 1840th or 1844th cysteine of enzyme active center of PI4KIII? (
[0145] It is obvious from the simulation that PAO forms two hydrogen bonds with the backbone N atom and the side chain S atom of CYS1844, wherein the hydrogen bond to N is also strong with a OH distance of 2.03 ?. In the dynamic simulation graph of the binding of PAO to wild type PI4KIII? (black) and PI4KIII? having mutation at the 1844.sup.th cysteine (red), the root mean square deviations of the protein scaffold with respect to the original structure of the two simulation trajectories tend to stabilize after 80 nanoseconds, wherein the structural change is greater in C1844S.
EXAMPLE 11: Virus Generation and Microinjection in Mice
[0146] Lentivirus was produced by invitrogen (Shanghai) using the BLOCK-iT? HiPerform? Lentiviral Pol II miR RNAi Expression System with EmGFP. Four miRNAs oligos targeting Efr3a were synthesized and inserted into pcDNA?6.2-GW/EmGFPmi vector. Knockdown efficiency was tested by RT-PCR or Western Blot method. Results showed that one of the vectors was most effective in knocking down Efr3a expression in HEK293T cells over-expressing EFR3a gene. Sequence of vector having the highest knockdown efficiency is AGGTATCATTCAGGTTCTGTT. The most effective miRNA vector was recombinated with pDONRTM221 and pLenti6/V5 DEST to generate the pLENT6/V5-GW/?EmGFP-miRNA vector via Gateway? recombination reactions. Lentivirus was generated by co-transfection of the pLENT6/V5-GW/?EmGFP -miRNA vector and Packaging Mix. Viral titer was obtained by serial dilutions in HEK293T cells. EGFP-positive cells were counted every 3 days. Knockdown efficiency was further obtained in lentivirus-transfected primary hippocampal neurons.
[0147] Male transgenic APP/PS1 mice (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax (MMRRC ID 034832-JAX) was maintained by crossing to C57BL mice. At 6 month-age, mice were anesthetized with 100 mg/kg Ketamine plus 20 mg/kg Xylazine and mounted ventral side down on stereotaxic apparatus with an electric blanket placed under its abdomen. Hair on the head was removed, skin was cut open, and a small hole was made through the skull. 2 ?L lentivirus solution (viral titer: 6?10.sup.?7) was injected with a syringe pump (Harvard Apparatus) within 20 min through a cannula system (external diameter, 0.29 mm, internal diameter, 0.1mm, RWD Life Science Co., Ltd) at 2.1 mm posterior to bregma, 2.3 mm lateral and 1.9 mm ventral. 5 min after injection, the needle was removed, skin was sutured, and the mouse was moved to 25? C. environment with supply of food and water. At 12 month-age, the mice were anesthetized again, and subjected to transcardiac perfusion using 4% para-formaldehyde (PFA) in PBS. Experiments complied with the policy of the Society for Neuroscience (USA) on the use of animals.
EXAMPLE 12
EFR3a Knockdown May Repair Atrophy of Neuronal Dendrites in APP/PS1 Mice, Tested by GFP Staining in Mouse Brain Slices
[0148] GFP staining in mouse brain slices: Brain slices (60 ?m thickness) were blocked with PBS-0.3% triton-5% BSA for 1 hr., followed by incubation with rabbit anti-GFP antibody (A11122, invitrogen, 1:100 dilution) overnight at 4? C. Then wash with PBS, incubate with biotinylated goat anti-rabbit IgG antibody (H+L) (AbboMax, Inc, 1:100 dilution) overnight at 4? C. PBS wash again, incubate with Cy3-Streptavidin (Jackson ImmunoResearch Laboratories Inc, 1:1000 dilution) for 2 hrs at RT. Images were taken under Zeiss LSM 511 confocal microscope, deconvoluted with AutoQuant X2, and analyzed with NeuronStudio software. The genotypes of brain slices and images of dendrites were blind to the imaging and analyzing personnel, respectively.
[0149] In
[0150] In
[0151] Mice and human possess two rbo homologs, EFR3a and EFR3b, both of which are enriched in the hippocampus and the like regions (Allen Brain Atlas) which are highly susceptible to AD. We investigated whether down-regulation of EFR3a gene can protect hippocampal neurons in APP/PS1 mice by using EGFP-tagged RNAi method. RNAi knockdown efficiency was shown in
EXAMPLE 13
PAO Improves Learning and Memory, Increases A?.SUB.42 .in CSF, but Decreases A?.SUB.42 .in Brain Membrane in APP/PS1 Mice, Detected by Composition Analysis of CSF and Membrane of Fractionated Mouse Brain
[0152] Cerebrospinal fluid collection: Mice were anesthetized with Ketamine and Xylazine, and were secured with head adaptors. The skin of the neck was shaved and cut open, the underlying subcutaneous tissue and muscles were separated laterally with forceps to expose the dura above the cisterna magna. A sharp-tipped glass capillary with a blunt end connected to a microinjection syringe was used to penetrate the dura. Following a noticeable change in resistance to the capillary tube insertion, the capillary entered the cisterna magna, CSF flowed into the capillary tube, until approximately 10-20 ?L was collected. The collected CSF was transferred into an Eppendorf tube containing protease inhibitor, and stored at ?80? C. until use.
[0153] Fractionation of mouse brain membrane: Obtain detergent-soluble A?.sub.42 through serial extraction. 5 times in volume of mouse brain hemisphere of Tris-buffered saline (TBS) was added for grounding and homogenizing, centrifuged at 100,000 g for 60 min and the supernatant was the TBS extract. The precipitate was collected, added 5 times in volume TBS containing 1% polyethylene glycol octylphenol ether for grounding, centrifuged and the supernatant was the TBS-Triton extract. The precipitate was collected again, added 5 times in volume TBS containing 1% SDS for grounding, centrifuged and the supernatant was the TBS-SDS extract. All three supernatants were collected, aliquoted and stored in ?80? C. refrigerator for ELISA assay.
[0154] The foregoing results in cultured cells and flies demonstrate that PAO facilitates A?.sub.42 secretion, reduces intraneuronal A?.sub.42 accumulation, and ameliorates synaptic failure, indicating that PAO is a potential drug for treating AD by facilitating A?.sub.42 secretion. To test this, we performed behavioral and biochemical experiments with APP/PSI mice gavaged with PAO at different doses.
[0155]
[0156] Having examined the toxicity of PAO gavage in wild-type mice as described in Example 8, the following experiments were performed with a concentration gradient of 0, 0.1, 0.3 and 1.0 mg/kg. 4 month-age APP/PSI mice and wild-type mice were subjected to PAO gavage, once a day every Monday through Friday for 6 consecutive weeks. Then, PAO administration was stopped for one week, and learning and memory abilities of mice were tested by the water maze experiment according to Vorhees and Williams. Compared to wild-type control, APP/PSI mice without PAO treatment exhibited impaired spatial learning and memory abilities (
EXAMPLE 14
PAO Gavage Ameliorates Cognitive Impairment of APP/PS1 Mice at Older Month-Age
[0157] In order to study the treatment effect of PAO on APP/PSI mice already having identifiable learning and memory dysfunction, 8 month-age APP/PSI mice and wild type littermates were subjected to PAO gavage at doses of 0 or 0.3 mg/kg body weight, once a day every Monday through Friday for 6 consecutive weeks. Then, administration was stopped for one week, and mice were subjected to novel object recognition test (NOR). The results showed that PAO treated mice spend significantly more time exploring novel object than old object that was explored the day before. In order to study effect of PAO on older month-age APP/PSI mice, as well as toxicity of prolonged administration on mice, 20 APP/PS1 mice at 18 month-age was randomly divided into two groups with 10 mice in each group. One group was subjected to PAO gavage at doses of 0.3 mg/kg body weight, once a day every Monday through Friday, the other group was subjected to water gavage; no administration at weekends. After 8 consecutive months, administration was stopped for one week, and mice were subjected to NOR test. During 8 months of gavage, two mice were dead in each group. NOR test showed that mice in PAO treatment group spend significantly more time exploring novel object than mice in the other group (
[0158] NOR test designing and
[0159] In
EXAMPLE 15
PI4KIII? Mutation Ameliorates Learning and Memory Defect in APP/PS1 Mice, Tested by Water Maze Experiment
[0160] Genetic down-regulation of PI4KIII? expression level or inhibition of its enzyme activity with PAO in A?.sub.42-expressing flies both ameliorates neural dysfunction; inhibition of PI4KIII? enzyme activity with PAO in APP/PSI mice also improves learning and memory abilities. In order to further clarify the role of PI4KIII? ion the neurodegeneration in APP/PS1 mice, the effect of heterozygous mutation of PI4KIII? (Pi4kaGt(RR0073)Byg/+. insertion of transposon pGT2Lxf into one copy of PI4KA gene may impede transcription of the gene copy, the resulted mRNA may only translate the truncated protein formed by the first ?265 amino acids on the N-terminus of PI4KIII? and the protein encoded by the reporter gene) on water maze experiment in APP/PS1 mice was examined. Pi4ka.sup.Gt(RROO73)Byg/+ mutation heterozygotes (MMRRC, Cat. #016351-UCD) were crossed with APP/PSI mice to obtain four groups of genotype mice: wild-type (WT), PI4KIII? mutation heterozygote (PI4K.sup.*/+), APP/PS1 (TG) and APP/PS1 with PI4KIII? heterozygous mutation (TG;PI4K.sup.*/+). When mice in the four groups reached 5 month-age, water maze experiments were performed according to Vorhees and Williams. As shown in
EXAMPLE 16
Down-Regulation of PI4KIII? Expression Level and Inhibition of PI4KIII? Enzyme Activity by PAO both Significantly Ameliorate Synaptic Transmission Plasticity Impairment in the Hippocampus of APP/PS1 Mice
[0161] In order to detect the effect of down-regulation of PI4KIII? expression level and inhibition of PI4KIII? enzyme activity by PAO on the synaptic transmission plasticity impairment in hippocampus of mouse, we induced and recorded long-term potentiation (LTP) of hippocampus CA3-CA1 synapses in mouse brain slices. 4 month-age APP/PS1 mice and wild type littermates were subjected to PAO gavage at doses of 0 or 0.3 mg/kg body weight, once a day every Monday through Friday for 4 consecutive weeks. Then, administration was stopped for one week. We found that the LTP amplitude of APP/PS1 mice without PAO treatment was significantly lower than the LTP amplitude of wild type mice and APP/PSI mice treated with PAO (
[0162] Method: Preparation of mouse hippocampal slices and recording of LTP of hippocampus CA3-CA1: Take a mouse of about 6 month-age, 30 g body weight, clean grade. Anesthetize the mouse by intraperitoneal injection of 1% pentobarbital sodium. Anesthetized to coma and surgery by thoracotomy to expose the entire heart; cut to open the right auricle; inject 30 mL of 4? C. PBS rapidly with a 50 mL syringe and a #3 needle through the left ventricle; decapitate; fix the head and cut with scissors along the skull midline and along both sides of the cranial base to open the skull, while continually washing the brain with ?0? C. anatomy fluid (the anatomy fluid is pre-filled with a mixed gas comprising 95% O.sub.2+5% CO.sub.2 for 30 min); then place the entire brain on a plate containing ?0? C. anatomy fluid; remove olfactory bulb and cerebellum with surgery knife and curved tweezers; separate the two cerebral hemispheres along the cranial line; after trimming brain block, add a small amount of ethyl a-cyanoacrylate glue to the base to ensure stably standing of the hippocampus tissue; rapidly place the base in the slicer tank containing ?0? C. anatomy fluid while continually filling of the mixed gas; cut sagittally to obtain brain slice with a thickness of 400 ?m; place the brain slice in a glass cup containing artificial cerebrospinal fluid (ACSF, pre-filled with a mixed gas for 30 min); incubate at room temperature (?25? C.) for 1?2 h before use. (ACSF in mM: 124 NaCl, 2 KCl, 2 MgSO.sub.4, 1.25 KH.sub.2PO.sub.4, 2 CaCl.sub.2, 26 NaHCO.sub.3, 10 D-glucose [pH 7.4], 300 mOsm).
[0163] Record field excitatory postsynaptic potentials (fEPSPs) at hippocampus CA1 region according to standard procedure: place two stimulating probes (FHC Inc., Bowdoin, ME) at the Schaffer collaterals of hippocampus CA3 region, place one glass microelectrode and one recording electrode at the stratum radiatum of the CA1 region. Distance between the stimulating probe and the recording probe is about 200?300 ?m. Stimulating intensity is 30-50% of the stimulating intensity to induce maximum amplitude of fEPSPs; stimulating frequency is 0.05 Hz. After the amplitude of induced fEPSPs was stabilized for 20 min, high frequency stimulation at 100 Hz was given through the stimulating probes and was repeated after 20 sec. After stimulation, continue with 0.05 Hz stimulation and record fEPSPs for 80 min. high frequency filter at 1 KHz, recording frequency at 20 kHz, pre-amplifier is Heka EPC 10 amplifier (Harvard Bioscience Inc., Ludwigshafen, HRB).
[0164]
EXAMPLE 17: Effects of PI, PI.SUB.4.P, PI.SUB.4,5.P on A?.SUB.42 .Aggregation/Oligomerization in Liposomes, Tested by Liposme Experiment
Liposme Experiment
[0165] 1) Dissolving synthetic A?.sub.42 with hexafluoro-2-propranol (HFIP) at a ratio of 1:1 (mg:mL), followed by 50 Hz sonication at RT for 15 min for homogenization. The sonicator is KUDOS ultrasonic instrument (Model SK250HP). [0166] 2) Dissolving the lipids (Table 1) with chloroform separately to prepare the lipids used for generating liposomes (Table 3) at a ratio of 1:1 (mg:mL), followed by 50 Hz sonication at RT for 15 min for homogenization. [0167] 3) Mixing the lipids according to the composition as listed Table 3, following by removing organic solvents from solution with lyophilizer. [0168] 4) Resuspending the precipitate after lyophilization with 1.0 mL Tris buffer (50 mM Tris, 120 mM NaCl, pH 7.0), followed by sonication in ice water for 30 min for homogenization. Stay at 4? C. for 48 hours for immunoblot analysis (primary antibody is anti-humanized A? monoclonal antibody 6E10, 1:1000 dilution). Before immunoblot analysis, 15 min of sonication is required for homogenization.
TABLE-US-00003 TABLE 3 Composition of liposome preparations Volume CAS # Mw (1 mg/ml) Mole % Phosphatidylserine 145849-32-7 757.95 8.9 68 ?L (PS) Phosphatidylcholine 63-89-8 734.04 15.4 121 ?L (PC) Phosphatidylethanolamine 1069-79-0 748.07 26.5 196 ?L (PE) Cholesterol (CH) 57-88-5 386.65 14.8 57 ?L Sphingomyelin (SM1) 85187-10-6 973.55 8.9 86 ?L Final conc. A?.sub.42 4514.04 1.0 ?M 4.5 ?L PI (diC16) 119943-95-2 833.01 20 ?M 17 ?L PI.sub.4P (diC16) 214332-61-3 956.96 20 ?M 19 ?L PI.sub.4,5P (diC16) 120595-88-2 1080.9 20 ?M 22 ?L
[0169] Effects of PI, PLAP, PIP2 on the aggregation/oligomerization of A?.sub.42 in liposome were analyzed and compared. In
EXAMPLE 18
Complex Formation of RBO/EFR3/EFR3A/EFR3B, PI4KIII?, and TTC7 on Cell Membrane
[0170] It is reported that yeast EFR3 protein forms a complex with PI4KIII? and a scaffold protein YYPI (referred to as TC7 in mammals, including two homologs TTC7A and TTC7B) on cell membrane and aggregates as PIK patches, and together regulate plasmalemma PI.sub.4P level and even PI4.5P level. YYPI interacts directly with N-terminus and central region of yeast PI4KIII? protein, thus playing an important role in construction and stabilization of PIK patches (Baird D, Stefan C, et al., 2008, J Cell Biol). Formation and function of PIK patches are also conservative in mammal cells (Nakatsu F, Baskin J F, et al., 2012, J Cell Biol). Drosophila homolog of TTC7 in flies is encoded by lethal (2) k14710 (I(2) k14710) gene.
[0171] To test the role of TTC7 protein in the neurodegeneration caused by intraneuronal A? accumulation, two transposon mediated transgenes were introduced into A?.sub.arc flies. P{lacW}1(2)k14710k.sup.15603 transposon (Bloomington Cat. #11134) was inserted into the first exon of l(2) k14710 gene in order to prevent transcription of l(2) k14710; the other one is P{EPgy2}bin3.sup.EY09582 (Bloomington Stock #20043). Four groups of flies were constructed in this experiment: control flies (ctrl), A?.sub.arc flies (A?.sub.arc), A?.sub.arc flies having one copy of P{lacW}1(2) k14710k.sup.15603 (A?.sub.arc-dttc7.sup.+/?, TTC7 down-regulating) and A?.sub.arc flies having one copy of P{EPgy2}bin3.sup.EY09582 (A?.sub.arc-dttc7-OE, TTC7 over-expressing). When adult flies of four groups reached age period of 5-10 day-age and 30-35 day-age, recording of EJP in GF pathway were conducted under 100 Hz brain stimulation.
[0172]
[0173] Preferred embodiments for carrying out the invention are described herein. The present invention is not limited to above embodiments. Any variation, modification, substitution, combination, and simplification without departing from the spirit and principle of the present invention belongs to equivalents of the present invention and is included within the scope of protection of the present invention.