Patent Application
20190263867
This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via EFS-Web as an ASCII text file entitled “0110-000572US01_ST25.txt” having a size of 4 kilobytes and created on Feb. 27, 2019. Due to the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR § 1.821(c) and the CRF required by § 1.821(e). The information contained in the Sequence Listing is incorporated by reference herein.
Post-translational modifications of the cytoskeletal protein tau are implicated in synaptic dysfunction in Alzheimer's disease and other tauopathies. Long-lasting synaptic plasticity underpinning learning and memory involves the insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into the postsynaptic membrane of dendritic spines and the removal of the receptors from spines. Under disease conditions, dendritic spines contain fewer AMPA receptors and elevated levels of modified, mislocalized tau. Proline-directed phosphorylation of serine (S) and threonine (T) residues in tau leads to postsynaptic dysfunction, but, at the time of the invention, details about cellular mechanisms remained unclear.
This disclosure describes identification of the residues and modifications of tau involved in the mislocalization of tau and the reduction of AMPA receptors in dendritic spines, and therapies designed to interfere with those modifications and the subsequent mislocalization of tau and/or the reduction of AMPA receptors. In some aspects, such therapies may be useful in subjects at risk of or exhibiting symptoms of Alzheimer's Disease, Parkinson's disease, chronic traumatic encephalopathy, and/or another tauopathy.
In one aspect, this disclosure provides peptides, compositions including those peptides, and methods of using those peptides and compositions. In some embodiments, the peptide preferably prevents the mislocalization of tau that leads to tau-mediated synaptic deficits.
In some embodiments, the peptide includes a tau peptide. In some embodiments, the peptide includes a protein transduction domain and/or a modification to increase the peptide's ability to cross the blood-brain barrier.
In some embodiments, the tau peptide includes a sequence of amino acids having at least 80% homology to SPVVSGDTS (SEQ ID NO:4). In some embodiments, the tau peptide includes a sequence of amino acids that includes at least one of SPVVSGDTS (SEQ ID NO:4) and APVVSGDTA (SEQ ID NO:5). In some embodiments, the tau peptide includes a sequence of amino acids that includes at least one of KSPVVSGDTSP (SEQ ID NO:6) and KAPVVSGDTAP (SEQ ID NO:7).
In some embodiments, the tau peptide includes at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, or at least 28 amino acids.
In some embodiments, the tau peptide includes DHGAEIVYKSPVVSGDTSPRHLSNVSST (SEQ ID NO:8). In some embodiments, the tau peptide includes DHGAEIVYKAPVVSGDTAPRHLSNVSST (SEQ ID NO: 9).
In some embodiments, the tau peptide includes a mutation that blocks the phosphorylation of at least one of S396 and S404 in tau. In some embodiments, at least one of S396 and S404 is replaced with an alanine.
In some embodiments, the protein transduction domain includes an HIV Trans-Activator of Transcription (TAT) domain. In some embodiments, the protein transduction domain includes GRKKRRQRRRPQ (SEQ ID NO: 10). In some embodiments, the protein transduction domain is conjugated to the N terminus of the tau peptide.
In some embodiments, the peptide reduces the localization of tau to the dendritic spines of a mechanically injured neuron by at least 10 percent.
In another aspect, this disclosure describes a method of making a peptide described herein.
In a further aspect, this disclosure describes a composition that includes a peptide described herein. In an additional aspect, this disclosure describes a virus encoding a peptide described herein.
In yet another aspect, this disclosure describes a method that includes administering a peptide, composition, or virus described herein. In some embodiments, the peptide may be administered via intraventricular injection or via intrathecal injection. In some embodiments, the virus may be introduced into a subarachnoid space.
In some embodiments, the method further includes administering a kinase inhibitor to the subject.
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure.
Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
In
This disclosure provides a peptide, compositions including the peptide, and methods of using the peptide and compositions. In some embodiments, the peptide prevents the mislocalization of tau that leads to tau-mediated synaptic deficits. In one embodiment, the peptide interferes with the phosphorylation of S396 and/or S404 in the C-terminal tail of tau. In some aspects, the peptides may be used as a therapy in subjects at risk of or exhibiting symptoms of Alzheimer's Disease, Parkinson's disease, chronic traumatic encephalopathy, and/or another tauopathy.
Post-translational modifications of the cytoskeletal protein tau are implicated in neurodegenerative diseases including Alzheimer's disease (AD) and other tauopathies including, for example, frontotemporal dementia with parkinsonism-17 (FTDP-17) and chronic traumatic encephalopathy (CTE). Tau is a multifunctional protein having an unstructured form that enables it to interact with many different proteins. At the time of the invention, however, little was known about how phosphorylation in different regions of tau related to postsynaptic dysfunction, and previous attempts to treat AD by blocking tau have had limited success.
Previous studies have shown tau mislocalization to dendritic spines plays a role in functional deficits (Hoover et al. 2010 Neuron 68(6):1067-1081; Ittner et al. 2010 Cell 142, 387-397), but the involvement of specific phosphorylation sites has not yet been defined. Example 1 describes a previously unreported pathway by which tau induces synaptic dysfunction in tauopathies by targeting specific phosphorylatable serine/phosphorylatable threonine (SP/TP) residues. As further described in Example 1, phosphorylation in two non-overlapping tau domains regulates a two-step process leading to postsynaptic dysfunction. First, the phosphorylation of S396 or S404 in the C-terminal tail of tau results in tau mislocalization to dendritic spines. Second, the phosphorylation of one or more residues in the proline-rich region of tau (the B domain) results in the decrease of AMPA receptors in the dendritic spines.
Example 3 describes a role for alpha-synuclein (αS) in tau missorting to dendritic spines and subsequent loss of postsynaptic AMPA receptors. In particular, A53T αS, a mutation of αS associated with familial Parkinson's disease, induces postsynaptic deficits that require GSK3β-dependent tau missorting to dendritic spines and calcineurin-dependent loss of postsynaptic surface AMPA receptors. As described in Example 3, when residues of tau were converted to unphosphorylatable residues, tau no longer mislocalized to dendritic spines even when A53T αS was expressed (se
The findings of Example 1 and Example 3 suggest that preventing either the mislocalization of tau or the reduction of AMPA receptors in dendritic spines including, for example, by targeting the specific post-translational modifications (e.g., phosphorylation) involved may provide promising therapies for tauopathies.
Thus, in one aspect, this disclosure provides peptides, compositions including those peptides, and methods of using those peptides and compositions to prevent the mislocalization of tau that leads to tau-mediated synaptic deficits.
In one aspect, this disclosure describes a peptide. In some embodiments, the peptide prevents the mislocalization of tau that leads to tau-mediated synaptic deficits. In some embodiments, the peptide reduces the localization of tau to dendritic spines of a mechanically injured neuron by at least 10 percent, at least 20 percent, at least 30 percent, at least 40 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, or at least 90 percent.
In one embodiment, the peptide interferes with the phosphorylation of S396 and/or S404 in the C-terminal tail of tau.
In some embodiments, the peptide includes a tau peptide. A tau peptide includes amino acids contained in the tau protein. In some embodiments, the tau peptide includes a peptide having at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology to the corresponding amino acids in the tau protein. In some embodiments, the tau peptide includes at least some of the amino acids of tau between positions S396 and S404. In some embodiments, the tau peptide includes a sequence of amino acids including each of the amino acids of tau between positions S396 and S404. For example, in some embodiments, the tau peptide includes PVVSGDT (SEQ ID NO:11). In some embodiments, the tau peptide includes a sequence having at least 70% homology to SPVVSGDTS (SEQ ID NO:4). In some embodiments, the tau peptide includes a peptide that includes amino acids of the tau protein except that the serine at least one of positions 369 and 404 of tau are replaced with an alanine. For example, in some embodiments, the tau peptide includes at least one of APVVSGDTS (SEQ ID NO: 12), SPVVSGDTA (SEQ ID NO: 13) and APVVSGDTA (SEQ ID NO:5).
In some embodiments, the tau protein is preferably human tau protein.
In some embodiments, the tau peptide includes at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, or at least 28 amino acids.
In some embodiments, the tau peptide includes up to 10 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 18 amino acids, up to 20 amino acids, up to 22 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 30 amino acid, up to 31 amino acids, up to 35 amino acids, up to 40 amino acids, up to 45 amino acids, up to 50 amino acids, or up to 100 amino acids.
In some embodiments, the tau peptide includes a sequence including SPVVSGDTS (SEQ ID NO:4); in some embodiments, the tau peptide includes a sequence including APVVSGDTA (SEQ ID NO:5). In some embodiments, the tau peptide the peptide includes a sequence including KSPVVSGDTSP (SEQ ID NO:6); in some embodiments, the tau peptide the peptide includes a sequence including KAPVVSGDTAP (SEQ ID NO:7).
In some embodiments, the tau peptide includes a sequence including DHGAEIVYKSPVVSGDTSPRHLSNVSST (SEQ ID NO:8). In some embodiments, the tau peptide includes a sequence having 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the sequence including DHGAEIVYKSPVVSGDTSPRHLSNVSST (SEQ ID NO:8). In some embodiments, the tau peptide includes a sequence consisting of DHGAEIVYKSPVVSGDTSPRHLSNVSST (SEQ ID NO:8).
In some embodiments, the tau peptide includes a mutation that blocks the phosphorylation of at least one of S396 and S404. In some embodiments, at least one of S396 and S404 is replaced with an alanine. In some embodiments, the tau peptide includes a sequence comprising DHGAEIVYKAPVVSGDTAPRHLSNVSST (SEQ ID NO: 9). In some embodiments, the tau peptide includes a sequence consisting of DHGAEIVYKAPVVSGDTAPRHLSNVSST (SEQ ID NO: 9).
In some embodiments, the peptide includes a protein transduction domain, that is, a sequence to make the peptide membrane permeable. In some embodiments, the peptide includes an HIV Trans-Activator of Transcription (TAT) protein transduction domain. In some embodiments an HIV Trans-Activator of Transcription (TAT) domain sequence can include GRKKRRQRRRPQ (SEQ ID NO: 10). In some embodiments, a protein transduction domain can include a cationic peptide sequence including, for example, a sequence including predominantly arginine, ornithine and/or lysine residues. In some embodiments, a protein transduction domain can include a hydrophobic sequence including, for example, a leader sequence. In some embodiments, the protein transduction domain is conjugated to the N-terminus of the tau peptide. In some embodiments, the protein transduction domain is conjugated to the C-terminus of the tau peptide. In some embodiments, a linker sequence may be included between a protein transduction domain and the tau peptide.
In some embodiments, the chemical structure of the peptide may be modified to increase specificity and/or blood-brain barrier crossing ability.
In some embodiments, including for example, when the peptide does not include a protein transduction domain, the peptide includes a modification to increase its ability to cross the blood-brain barrier. For example, the peptide may be conjugated to a blood-brain barrier shuttle. (Malakoutikhah et al. 2011 Angew Chem Int Ed Engl. 50(35):7998-8014.) In some embodiments, the structure of the peptide may be altered to increase its chemical stability. Modifications may include, for example, modification of peptide bonds, introduction of nonproteinogenic amino acids, and/or modification of the amino acid side chains and/or terminal residues. (See, for example, Peptide Modifications to Increase Metabolic Stability and Activity, Cudic (ed.), Humana Press (2013).) Exemplary embodiments of peptides that prevent the mislocalization of Tau are described in
In another aspect, this disclosure describes methods of making the peptides described herein. The peptide may be synthesized by any suitable method. For example, in some embodiments, the peptide may be chemically synthesized using a solid phase peptide synthesis (SPPS) technique by incorporating amino acids into a peptide of any desired sequence.
In some embodiments, the peptide may be biologically expressed by an appropriate vector (plasmid or virus).
In a further aspect, this disclosure describes compositions including a peptide described herein. The compositions may be suitable for oral, rectal, vaginal, topical, nasal, ophthalmic or parenteral (including subcutaneous, intramuscular, intraperitoneal, and intravenous) administration.
A composition may also include, for example, buffering agents to help to maintain the pH in an acceptable range or preservatives to retard microbial growth. A composition may also include, for example, carriers, excipients, stabilizers, chelators, salts, or antimicrobial agents. Acceptable carriers, excipients, stabilizers, chelators, salts, preservatives, buffering agents, or antimicrobial agents, include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives, such as sodium azide, octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; polypeptides; proteins, such as serum albumin, gelatin, or non-specific immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG).
The composition may be presented in unit dosage form and can be prepared by any of the methods well-known in the art of pharmacy. In some embodiments, a method includes the step of bringing the peptide into association with a pharmaceutical carrier. In general, a composition may be prepared by uniformly and intimately bringing the peptide into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into a desired formulation.
Methods of Administration and/or Treatment
A peptide described herein may be administered to a subject alone or in a pharmaceutical composition. In some embodiments, the peptide may be administered to a subject by introducing a vector (for example, a virus or plasmid) encoding the peptide into the subject. In some embodiments, the vector may be an adenovirus. The peptide or vector may be delivered using any suitable method. The subject may be an animal or a human. In some embodiments, the subject may be at risk of or may exhibit symptoms of Alzheimer's Disease, Parkinson's disease, chronic traumatic encephalopathy, and/or another tauopathy.
The peptide, a composition including the peptide, or a vector encoding the peptide can be administered to a vertebrate, more preferably a mammal, such as a human patient, in an amount effective to produce the desired effect. A peptide, a composition including the peptide, or a vector encoding the peptide can be administered in a variety of routes, including orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form.
A formulation can be administered as a single dose or in multiple doses. Useful dosages of a peptide, a composition including the peptide, or a vector encoding the peptide can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art.
In some embodiments, the peptide may preferably be injected. For example, the peptide may be injected into the brain including, for example, into a ventricle. In some embodiments, the peptide may preferably be introduced via intrathecal injection.
In some embodiments, including, for example, when a vector encoding the peptide is administered, the peptide may preferably be administered by injection into a subarachnoid space.
In some embodiments, including, for example, to treat traumatic brain injuries, the peptides may be transfused into the cerebrospinal ventricular system.
In some embodiments, a peptide, a composition including the peptide, or a vector encoding the peptide may be administered to a subject in combination with a kinase inhibitor. For example, a kinase inhibitor may include at least one of a calcineurin inhibitor including, for example, cyclosporin, voclosporin, pimecrolimus and tacrolimus (FK506); a cdk5 inhibitor; and a gskβ inhibitor including, for example, tideglusib. The kinase inhibitor may be administered by any suitable method.
Dosage of a peptide, a composition including the peptide, or a vector encoding the peptide may be varied so as to obtain an amount of the active agent which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of factors including, for example, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the aurone, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art may determine and prescribe the effective amount of the pharmaceutical composition, peptide, or a vector encoding the peptide required.
A peptide, a pharmaceutical composition including the peptide, or a vector encoding the peptide may be used to treat or prevent a tauopathy. Exemplary tauopathies include but are not limited to Alzheimer's Disease, A53T α-synuclein-associated familial Parkinson's disease, traumatic brain injury (TBI), and other diseases that include tau missorting.
In some embodiments, this disclosure provides a therapeutic method of treating a subject suffering from a tauopathy by administering a peptide, a pharmaceutical composition including the peptide, or a vector encoding the peptide to the subject. Therapeutic treatment is initiated after diagnosis or the development of symptoms of tauopathy.
In some embodiments, this disclosure provides a method of treating a subject prophylactically, to prevent or delay the development of a tauopathy. Treatment that is prophylactic, for instance, can be initiated before a subject manifests symptoms of a tauopathy. An example of a subject that is at particular risk of developing a tauopathy is a person who has suffered traumatic brain injury (TBI) or a person having a A53T mutation in α-synuclein, which causes familial Parkinson's disease. Treatment may be performed before, during, or after the diagnosis or development of symptoms of a tauopathy. Treatment initiated after the development of symptoms may result in decreasing the severity of the symptoms, or completely removing the symptoms.
Administration of the peptide, a pharmaceutical composition including the peptide, or a vector encoding the peptide to the subject can occur before, during, and/or after other treatments. The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
This Example describes the characterization of phosphorylation in two non-overlapping tau domains that can regulate a two-step process leading to postsynaptic dysfunction. First, tau mislocalizes to dendritic spines, and this process depends on the phosphorylation of S396 or S404 in the C-terminal tail of tau. Second, AMPA receptors in the spines are diminished, a reduction that involves both the mislocalization of tau and the phosphorylation of one or more of five SP/TP residues (S202, T205, T212, T217, and T231) in the proline-rich region of tau.
All common chemical reagents and cell culture supplies were purchased from Sigma-Aldrich (St. Louis, Mo.), Promega (Madison, Wis.), and Thermo-Fisher Scientific/Invitrogen/Life Technologies (Waltham, Mass.) unless otherwise indicated.
All human tau and dsRed constructs were expressed in the pRK5 vector and driven by the cytomegalovirus promotor (Takara Bio USA (formerly known as Clontech Laboratories, Inc.), Mountain View, Calif.). All human tau was n-terminally fused to enhanced GFP (eGFP). The wild type, native human tau construct encoded human four-repeat tau lacking the transcriptional-variant n-terminal sequences (0N4R) and contained exons 1, 4, 5, 7, 9-13, 14 and intron 13. P301L mutant as well as alanine and glutamate tau variant constructs were created using step-wise site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, Calif.). PCR primers for mutagenesis were 15-22 nucleotides long, centered on mutated nucleotide(s) (Integrated DNA Technologies, Coralville, Iowa). All nucleotide mutations as well as plasmid construct integrity were confirmed with Senger Sequencing (University of Minnesota Genomics Center, Minneapolis, Minn.). Δtau314 constructs were generated and confirmed as discussed in Zhao, X. et al. 2016 Nat. Med. 22, 1268-1276. Tau sequence numbering was based on the longest functional human isoform: 441-tau (2N4R tau; NCBI reference sequence: NP_005901.2).
Dissociated rat primary hippocampal neuron cultures described in this study were conducted in accordance with the American Association for the Accreditation of Laboratory Animal Care and Institutional Animal Care and Use Committee at the University of Minnesota (protocol #1211A23505). Briefly, a 25 mm diameter glass coverslip (0.08 mm thickness) was silicone-sealant-fastened to the bottom of a 35 mm culture dish with a bored hole and sterilized. Coverslips were coated with poly-D-lysine. Hippocampi were dissected from CO2-anesthetized neonatal Sprague-Dawley timed-pregnancy rats (Envigo Corporation, Huntingdon, UK) at 0-24 hours of life. Hippocampi were enzymatically digested in Earle's Balance Salt Solution (EBSS) supplemented with 1% glucose and cysteine-activated papain. Digestion was blocked with dilute DNase, and cells were rinsed in fresh EBSS and plated in plating medium (minimal essential medium with Earle's salts, 10% fetal bovine serum, 2 mM glutamine, 10 mM sodium pyruvate, 10 mM HEPES, 0.6% glucose, 100 U/ml penicillin and 100 mg/mL streptomyocin) at 1.0×106 cells/dish. After 18 hours, cell adherence was established. Cells were then grown in neurobasal medium (NbActiv1; BrainBits LLC, Springfield, Ill.) and incubated at 37° C. in a 5% CO2 biological incubator.
After 5-7 days in vitro (DIV), cells were transfected. DNA plasmid transfection was performed using standard calcium phosphate precipitation and incubation. Briefly, neurons were transfected with human tau constructs and dsRed (2:1 by plasmid DNA mass) for imaging experiments, and with human tau alone for electrophysiology. Precipitated DNA was applied to cells in a solution of glial conditioned medium (neurobasal medium previously conditioned for 14 days on a glial monolayer and reserved) containing 100 μM APV to prevent calcium-toxicity. After 3-4 hours transfection time, cells were rinsed in glial conditioned medium and grown in neurobasal medium as described above until mature (21-28 total DIV).
Miniature EPSCs were recorded from cultured dissociated rat hippocampal neurons at 21-25 DIV with a glass pipette (resistance ˜5 MΩ) at holding potentials of −65 mV on an Axopatch 200B amplifier (output gain=1; filtered at 1 kHz; made by Molecular Devices, San Jose, Calif.). Input and series resistances were assessed before and after recording mEPSCs (5-20 minutes) and found to have no significant difference before and after recording. Recording sweeps lasted 200 ms and were sampled for every 1 s (pCLAMP; Molecular Devices, San Jose, Calif.). Neurons were bathed in bubble-oxygenated artificial cerebral spinal fluid (ACSF) at 23° C. with 100 μM APV (NMDAR antagonist), 1 μM TTX (sodium channel blocker), and 100 μM picrotoxin (GABAa receptor antagonist). Passive oxygen perfusion was established with medical-grade 95% O2-5% CO2. ACSF contained (in mM) 119 NaCl, 2.5 KCl, 5.0 CaCl2, 2.5 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 11 D-glucose. The internal solution of the glass pipettes contained (in mM) 100 cesium gluconate, 0.2 EGTA, 0.5 MgCl2, 2 ATP, 0.3 GTP, and 40 HEPES. The pH of internal solution was normalized to 7.2 with cesium hydroxide and diluted to a trace osmotic deficit in comparison to ACSF (˜300 mOsm). All analysis of recordings was performed manually using MiniAnalysis (Synaptosoft Inc., Fort Lee, N.J.). Minimum parameters were set at greater than 1 min stable recording, and event amplitude greater than 2 pA. A mEPSC event was identified by distinct fast-rising depolarization and slow-decaying repolarization. Combined individual events were used to form relative cumulative frequency curves, whereas the means of all events from individual recordings were treated as single samples for further statistical analysis.
Transfected cells were continually bathed in neurobasal media and were passively perfused with medical-grade 95% O2-5% CO2. Micrographs were taken on a Nikon epifluorescent inverted microscope with 60× oil lens with a computerized focus motor at DIV 21-23. All digital images were processed using METAMORPH Imaging System (Universal Imaging Corporation, Molecular Devices, San Jose, Calif.). Images were taken as 15 plane stacks at 0.5 micron increments, processed by deconvolution to the nearest planes, and averaged against other stacked images. A dendritic spine was defined as having an expanded head diameter, greater than 50% larger in diameter than the neck. The number of spines per neuron were counted and normalized to a 100 m length of dendritic shaft.
Roscovitine and CHIR99021 were purchased from Sigma-Aldrich (St. Louis, Mo.) and were diluted in DMSO to four 1000× concentration aliquots for ultimate concentrations in neurobasal medium of 0.05, 0.5, 5 and 10 M. Primary cultured rat hippocampal neurons transfected with dsRed and eGFP-P301L-tau were treated with one of four 1000× aliquots or DMSO vehicle on DIV 20-22. Treated cells were incubated for 24 hours prior to imaging on DIV 21-23. Cell death was visually assessed under differential interference contrast (DIC) for decreased cell density, lost soma adhesion, gross qualitative neurite retraction. If evidence of cell death was observed under DIC, cells were fixed in 4% sucrose and 4% paraformaldehyde in PBS and stained with 300 nM DAPI in PBS. DAPI stained cells were analyzed under fluorescent microscopy for nuclear pyknosis and karyorrhexis; dsRed expressing cells were analyzed for spine loss. If no cell death was evident under DIC, live fluorescent images were acquired and analyzed as above.
All statistics were performed in Prism 6 (Graphpad Software, San Diego, Calif.). One- and two-way ANOVA was used for univariate and two-variable analysis respectively. If ANOVA revealed significant variance between all groups, post-hoc analysis was performed using Bonferroni analysis adjusted for multiple groups. Univariate cumulative frequency distributions were compared using the unmodified Kolmogorov-Smirnov goodness of fit test. For all, statistical significance was set for α=0.05.
To test if differential phosphorylation of distinct tau domains impairs postsynaptic function, three domains (referred as A-, B- and C-domains) of tau were formulated in a semi-random fashion, each containing clusters of four or five SP/TP residues (
As used in this Example, a “P301L mutant” refers to tau with the P301L mutation, a mutation linked to frontotemporal dementia with parkinsonism linked to chromosome 17, and “wild type” refers to tau without the P301L mutation. As used herein, “variant” refers to tau with SP/TP substitutions, and “native” refers to tau without SP/TP substitutions.
To identify the phosphorylation residues that regulate the mislocalization of tau, the effect on the subcellular distribution of tau of blocking phosphorylation in each of the three domains was tested. At 7-10 days in vitro, dsRed (to visualize cellular morphology) and P301L mutant or wild type eGFP-tau constructs with alanine substitutions in the A-domain (A-Ala), B-domain (B-Ala) or C-domain (C-Ala) were co-expressed in cultured rat hippocampal neurons. At 21 days in vitro (DIV), the dendrites of live neurons were photographed and the percentage of spines containing eGFP-tau was determined. eGFP-tau was found to be distributed throughout the dendritic shaft in all conditions, and significantly more eGFP-containing spines were observed in neurons expressing P301L mutant eGFP-tau (
Additional support for this conclusion was obtained by evaluating the effects of differential phosphorylation in each domain using tau variants with glutamate mutations, which mimic phosphorylation by increasing the negative charge. Because pseudophosphorylation of the A-residues is neither necessary nor sufficient to mediate tau-induced deficits (
The results described above demonstrate a role for phosphorylation on the mislocalization of tau to the dendritic spine, a glutamatergic postsynaptic compartment. Next, the role of phosphorylation in postsynaptic function was evaluated by testing the effect of blocking phosphorylation on AMPA receptor function. A-Ala, B-Ala and C-Ala variants of P301L mutant and wild type eGFP-tau were expressed in cultured rat hippocampal neurons, and whole-cell, patch-clamp electrophysiology was performed to record glutamatergic mini-excitatory postsynaptic currents (mEPSCs). In neurons expressing P301L mutant eGFP-tau, mEPSCs with smaller amplitudes (black lines and symbols,
To further test the hypothesis that the phosphorylation within the B- and C-domains collaborates to disrupt postsynaptic function, the effects of B-Glu, C-Glu and B+C-Glu on tau-induced glutamatergic postsynaptic function were examined by measuring mEPSCs in cultured rat hippocampal neurons expressing P301L mutant or wild type eGFP-tau. As expected, neurons expressing P301L mutant eGFP-tau showed reductions in mEPSC amplitudes, irrespective of phosphomimetic mutations (
To refine the identification of C-residues responsible for mislocalization, phosphorylation of specific SP/TP residues in the C-domain of P301L mutant eGFP-tau was blocked by mutating those residues to alanine (
There are 85 putative phosphorylation residues in tau, which vary in their extent of phosphorylation. Using a mass-spectrometry-based assay to measure the stoichiometry of phosphorylated residues in soluble wild type tau expressed in Sf9 insect cells and human neuronal iPSCs, the most frequently phosphorylated SP/TP residues were found to be S199, S202, T205, T212, T217, T231, S235, S396 and S404. Specifically, ˜85% of the wild-type-tryptic fragments containing S396 and S404 were modified (expressed as ˜15% unmodified), indicating that one or both residues are phosphorylated in ˜85% of wild type tau molecules expressed. If the stoichiometry of phosphorylation at these two residues is as high under “normal” physiological conditions, then most wild type tau proteins would be mislocalized in dendritic spines, which contradicts previous findings. This discrepancy suggests that the stoichiometry of phosphorylation in primary neurons and the brain may differ from that in the insect and iPSC culture paradigms, or that one or more “bottlenecks” or rate-limiting steps exist in the pathway leading to tau mislocalization. For example, it has been previously reported that the truncation of tau at D314 is also required for tau mislocalization (Zhao et al. 2016 Nat. Med. 22, 1268-1276).
This Example shows that postsynaptic dysfunction is the result of a coordinated progression of differential phosphorylation and cleavage, as depicted in the conceptual model of
The exact upstream factors causing cellular stress leading to the pathological activation of proteases and kinases are unknown. The unfolded protein response that is activated by endoplasmic reticulum stress (ER stress) (Su et al. 2016 Nat. Cell Biol. 18, 527-539) increases phosphorylation of the S202 and S205 residues in the B-domain (Kim et al. 2017 PLoS Genet. 13, 1-22). ER stress activates gsk3β (Liu et al. 2016 Mol. Neurobiol. 35, 983-994) and caspase-2 (Uchibayashi et al. 2011 J. Neurosci. Res. 89, 1783-1794).
The dysregulation of cdk5, which phosphorylates the C-site S404 of tau, and gsk3β, which phosphorylates the C-site S396 of tau, have been previously implicated in the pathogenesis of Alzheimer's disease. A combination of gsk3β and cdk5 inhibitors was needed to block tau-mediated synaptic changes, offering a potential explanation for the failure of tideglusib, a gsk3β inhibitor, in a recent clinical trial (Lovestone et al. 2015 J. Alzheimers Dis. 45, 75-88).
It may be of interest to delineate the downstream events leading to a reduction in postsynaptic AMPA receptors. One possibility is the internalization of GluA1 subunits of AMPA receptors through dephosphorylation by calcineurin, which interacts with a segment in the proline-rich domain (aa 198-244) encompassing the B-domain (S202-T231;
As shown in Example 1, phosphorylation of the C-domain drives tau-missorting whereas the B-domain drives subsequent loss of AMPA receptors (AMPARs) caused by tau. Without wishing to be bound by theory, it is believed that that Alzheimer's disease (AD) and/or traumatic brain injury (TBI) activate glycogen synthase kinase 3 beta (gsk3β) and cyclin-dependent kinase 5 (cdk5) which phosphorylate the C-domain of tau, driving tau to spines, resulting in further phosphorylation of the B-domain, causing loss of AMPARs (see
Two peptides were synthesized to block the initial step of the cellular cascade that leads to tau-mediated synaptic deficits:
An additional peptide was synthesized and tested but did not block the cellular cascade that leads to tau-mediated synaptic deficits:
The majority (>60%) of frontotemporal dementia with parkinsonism-17 (FTDP-17) is caused by three mutations: P301L/S, N279K and “10+16”. Cells and animals expressing a P301L mutant tau are frequently used as tauopathy models (Snowden et al. 2002 Br J Psychiatry 180:140-143). In neurons expressing P301L mutant tau proteins, both WT peptide and AP peptide blocked tau mis-sorting, and the AP peptide was observed to have a stronger effect. In contrast, the EP peptide did not block tau mis-sorting. Results are shown in
In another tauopathy model, the AP peptide was observed to block tau mislocalization caused by Aβ oligomers, which are believed to be the key initiator of neural deficits in AD. Results are shown in
In a tauopathy model for traumatic brain injuries (TBI) based on the model of Hemphill et al. 2011 PLoS One. 6(7):e2289), mechanical injuries to neurons induce tau missorting to dendritic spines (
This Example shows that A53T α-synuclein, which is associated with familial Parkinson's disease, induces phosphorylation-dependent tau mislocalization to dendritic spines and associated postsynaptic deficits.
Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease. Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. As further described in this Example, synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice expressing human wild-type, A53T, and A30P α-synuclein was analyzed. Increased α-synuclein expression was found to lead to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein were found to exhibit postsynaptic dysfunction including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. Mechanistically, postsynaptic dysfunction requires GSK3β-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. This tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial Parkinson's disease cases as well as some subgroups of Parkinson's disease cases with extensive tau pathology.
Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease. It is characterized by both motor symptoms and the convergence of alpha-synuclein (αS), tau, and amyloid-β pathology. Sporadic PD is clinically heterogeneous. Genetic abnormalities including αS gene (SNCA) amplification, as well as A53T, A30P, and E46K αS point mutations are linked to familial PD. These inherited forms of PD are also heterogeneous. Each features a different time of onset, clinical presentation and histopathology (Petrucci et al. 2015 Parkinsonism Relat. Disord. 22 Supl: S16-22). Particularly, tau and αS pathologies frequently coexist in the Contursi kindred who carry the A53T αS point mutation (Duda et al. 2002 Acta Neuropathol. 104(1):7-11). This Example describes the exploration of whether A53T mutant αS activates additional signaling pathways that are distinct from those activated by wild-type (WT) αS and other mutants. αS is a cytosolic protein that is enriched in the presynaptic terminals of neurons and can associate with the plasma membrane. To identify the cellular mechanisms underlying the clinical and pathological diversity of PD, changes in the synaptic function of neurons expressing multiple αS variants were compared. This Example describes the finding that A53T αS induces postsynaptic deficits that require GSK3β-dependent tau missorting to dendritic spines and calcineurin-dependent loss of postsynaptic surface AMPA receptors.
All common reagents used in this Example were purchased from Sigma Aldrich (St. Louis, Mo.) unless otherwise noted.
The four transgenic mouse lines used in the present study are listed in
Hippocampi from Tg mice were suspended and mechanically homogenized in 10 volumes of ice-cold TNE Buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, complete Mini Protease Inhibitor Cocktail, and Phosphatase Inhibitor Cocktails 2 and 3—inhibitors 1:100, Sigma Aldrich, St. Louis, Mo.) in a polystyrene tube. Homogenized tissue was aliquoted and diluted with equal volumes of ice-cold Complete TNE (TNE, 1% sodium dodecyl sulfate, 0.5% Nonidet P-40, 0.5% sodium deoxycholate). Estimation of protein concentration for protein correction and dilution was performed utilizing the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, Mass.).
Concentration-corrected protein samples were diluted in reducing sample buffer (Boston BioProducts, Ashland, Mass.), electrophoresed on 4-20% Criterion TGX gels (Bio-Rad Laboratories, Hercules, Calif.) and transferred onto Amersham 0.45 μm nitrocellulose membranes (GE Healthcare, Chicago, Ill.). Membranes were probed with primary antibodies of total αS (Catalog No. 610787, BD Biosciences, San Jose, Calif.), human αS (HuSyn1; Lee et al. 2002 PNAS 99, 8968-8973), and α-tubulin (Catalog No. ab4074, Abcam, Cambridge, UK) and visualized utilizing enhanced chemiluminescent reagents (Thermo Fisher Scientific, Waltham, Mass.) via ImageQuant LAS 4000 detection system (GE Healthcare, Chicago, Ill.). Densitometry analysis was performed utilizing ImageQuant TL 8.1 software (GE Healthcare, Chicago, Ill.).
Acute coronal hippocampal slices (350 μm thick) were obtained from 3-6 month old non-transgenic (TgNg) and Tg mice from lines 12-2 (WT), H5 (A53T), G2-3 (A53T), and O2 (A30P). Slices were kept in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 124, KCl 5, NaH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2 and glucose 10, gassed with 95% O2/5% CO2 (pH=7.3-7.4). Slices were incubated in ACSF at room temperature for at least 1 hour before use and then they were transferred to an immersion recording chamber, superfused at 2 mL/minute with gassed ACSF and visualized under an Olympus BX50WI microscope (Olympus Optical, Japan). Picrotoxin (50 μM) and CGP54626 (1 μM) were added to the solution to block GABAa and GABAb receptors respectively. Whole-cell electrophysiological recordings were obtained from CA1 pyramidal neurons. Patch electrodes (3-10 MΩ) were filled with internal solution containing (in mM): cesium-gluconate 117, HEPES 20, EGTA 0.4 NaCl 2.8, TEA-Cl 5, ATP-Mg+2 2, GTP-Na+0.3 (pH=7.3). Recordings were obtained with PC-ONE amplifiers (Dagan Instruments, Minneapolis, Minn.). Membrane potential was held at −70 mV. Signals were filtered at 1 kHz and acquired at 10 kHz sampling rate and fed to a Pentium-based PC through a DigiData 1440A interface board. The pCLAMP 10.4 (Axon Instruments, Molecular Devices, San Jose, Calif.) software was used for stimulus generation, data display, acquisition and storage. To record evoked excitatory postsynaptic currents (EPSCs), theta capillaries filled with ACSF were used for bipolar stimulation and placed in the stratum radiatum to stimulate Schaffer collaterals (SC). Input-output curves of EPSCs were made by increasing stimulus intensities from 20 μA to 80 μA. Paired pulses (2 ms duration) were applied in the SC with 25 ms, 50 ms, 75 ms, 100 ms, 200 ms, 300 ms, and 500 ms interpulse intervals and the paired-pulse ratio was calculated (PPR=2nd EPSC/1st EPSC). Synaptic fatigue was assessed with 15 consecutive stimuli with 25 ms interval. AMPA currents were obtained at a holding potential of −70 mV and NMDA currents at +30 mV. To ascertain the AMPA to NMDA receptor current ratio the NMDA component was measured 50 ms after the stimulus, when the AMPA component had decayed. For long-term potentiation (LTP) induction a tetanic stimulation (4 trains at 100 Hz for 1 second; 30 second intervals) was applied in the SC. EPSC amplitude was normalized to 10 min of baseline recording. After LTP induction, neurons were recorded for 45 minutes. The presence of LTP was determined by comparing the last 5 minutes of baseline with the last 5 minutes of recording. For miniature EPSC recordings TTX (1 μM) was also included in the solution. Normality was verified with a Kolmogorov-Smirnov test in analyses of cumulative curves and groups were compared using a one-way ANOVA with Fisher LSD post-hoc analysis. When data did not meet normality, a one-way Kruskal-Wallis test with Dunn's method post-hoc was applied.
All eGFP, tau, αS and DsRed constructs were expressed in the pRK5 vector and driven by a cytomegalovirus promoter (Takara Bio USA (formerly known as Clontech Laboratories, Inc.), Mountain View, Calif.). All tau and αS constructs were tagged with eGFP on the N-terminus. The WT tau construct encodes human four-repeat tau lacking the N-terminal sequences (0N4R) and contained exons 1, 4 and 5, 7, and 9-13, intron 13, and exon 14. Using WT tau as a template, QuickChange site-directed mutagenesis (Agilent Technologies, Santa Clara, Calif.) was used to generate two tau constructs termed AP tau and E14 tau. All 14 S/P or T/P amino acid residues (T111, T153, T175, T181, S199, 5202, T205, T212, T217, T231, S235, S396, S404, and S422) were mutated to alanine (AP) or glutamate (E14). Numbering is based on the longest (2N4R) 441-amino acid adult brain isoform of human tau. All tau constructs were characterized in Hoover et al. 2010 Neuron 68:1067-1081. Site directed mutagenesis was used to generate A30P, E46K, and A53T αS from WT αS. All sequences were confirmed with Sanger Sequencing (UMN Genomics, Minneapolis, Minn.).
A 25 mm glass polylysine-coated coverslip (thickness, 0.08 mm) was glued to the bottom of a 35 mm culture dish with a 22 mm hole using silicone sealant as previously described (Lin et al. 2004 Biochem Biophys Res Commun. 316(2):501-11). Dissociated neuronal cultures from mouse and rat hippocampi at postnatal day one were prepared as previously described (Hoover et al. 2010 Neuron 68:1067-1081). Briefly, hippocampi were dissected and stored in ice-cold Earl's Balance medium supplemented with 1 mM D-glucose. Rat hippocampal neurons from each litter were pooled before plating; whereas mouse hippocampal neurons were separated by pup before plating. Neurons were plated onto prepared 35 mm culture dishes at a density of 1×106 cells per dish. The age of cultured neurons was counted from the day of plating as one day in vitro (DIV). All experiments were performed on neurons from at least 3 independent cultures. Neurons at 6-8 DIV were transfected with appropriate plasmids using the standard calcium phosphate precipitation method as previously described (Liao et al. 2005 PNAS. 102(5): 1725-30). After transfection, neurons were placed in a tissue culture incubator (37° C., 5% CO2) and allowed to mature and develop until three weeks in vitro, a time at which neurons express high numbers of dendritic spines with mature morphologies. Mouse culture genotype was ascertained by Northern blot and reverse transcription-PCR analysis of ex-vivo tail clippings (as described above).
To detect the distribution of endogenous synaptic proteins with high resolution, low-density neuronal cultures were prepared as previously described with some modifications (Lin et al. 2009 Neuropsychopharmacology 34(9):2097-111). Dissociated neuronal cultures from Tg mouse hippocampi at postnatal days 1-2 were plated into 12-well culture plates at a density of 50,000-100,000 cells per well. Each well contained a polylysine-coated 12 mm glass coverslip. The 12 mm coverslips with 7 DIV low-density cultured neurons were transferred to high-density neuronal cultures in 60 mm dishes (4 coverslips per dish; to encourage survival).
Miniature excitatory postsynaptic currents (mEPSC) were recorded from cultured dissociated rat hippocampal neurons at 21-25 DIV with a glass pipette (resistance of ˜5 MΩ) at holding potentials of −55 mV and filtered at 1 kHz with an output gain, a, of 0.5 (mouse culture) and 1 (rat culture) as previously described (Miller et al. 2014 Euro. J. Neurosci. 39: 1214-1224). Briefly, neurons were bathed in artificial cerebrospinal fluid (ACSF) at room temperature (25° C.) with 100 μM APV (an NMDAR antagonist), 1 μM TTX (a sodium channel blocker), and 100 μM picrotoxin (GABAa receptor antagonist), gassed with 95% O2-5% CO2. The ACSF contained (in mM): 119 NaCl, 2.5 KCl, 5.0 CaCl2, 2.5 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 11 glucose. The internal solution in the patch pipette contained (in mM) 100 cesium gluconate, 0.2 EGTA, 0.5 MgCl2, 2 ATP, 0.3 GTP, and 40 HEPES (pH 7.2 with cesium hydroxide). mEPSC traces were recorded using Axopatch 200B amplifier and pClamp 11 (Molecular Devices, San Jose, Calif.). Recordings ranged from 5-20 minutes and stable traces longer than 2 minutes in duration were analyzed. All mEPSCs>3 pA were manually counted with MiniAnalysis (Synaptosoft Inc., Fort Lee, N.J.). Each mEPSC event was visually inspected and only events with a distinctly fast-rising phase and a slow-decaying phase were accepted. Relative cumulative frequencies were derived from individual events and the averaged parameters from each neuron were treated as single samples in any further statistical analyses.
The 35 mm culture dishes fit tightly in a custom holding chamber on a fixed platform above an inverted Nikon microscope sitting on a B
Cultured neurons were fixed and permeabilized successively with 4% paraformaldehyde, 100% methanol, and 0.2% Triton X-100 (Lin et al. 2009 Neuropsychopharmacology 34(9):2097-111). For all immunocytochemical staining, primary antibodies were diluted at 1:50 or 1:100 in 10% donkey serum in PBS and rhodamine (red)- or FITC (green)-labeled secondary antibodies were diluted at 1:100 or 1:200 respectively. Mouse anti-synaptophysin (Thermo Fisher Scientific, Waltham, Mass.) antibodies (1:100 dilution) were used to detect presynaptic terminals. Commercial antibodies against PSD-95 were used as a postsynaptic marker to stain dendritic spines (rabbit polyclonal, Invitrogen, Carlsbad, Calif.; mouse monoclonal, Millipore, Burlington, Mass.; 1:100 dilution) as previously described (Lin et al. 2009 Neuropsychopharmacology 34(9):2097-111). The rabbit polyclonal antibodies against the N-terminus of GluA1 subunits were generous gifts from Dr. Richard Huganir (Johns Hopkins University Medical School). The fixed neurons were incubated with primary antibodies at 4° C. overnight and subsequently incubated with secondary antibodies for 1-2 hours at room temperature (PSD-95) or a 37° C. incubator (synaptophysin). The fluorescent images of antibody staining and transfected exogenous proteins (eGFP-αS or eGFP alone) were taken with an inverted Nikon microscope (see In vitro Neuronal Imaging and Analysis above). In
Spatial learning and memory was evaluated using the Barnes Maze as previously described with some modifications (protocol available on the world wide web at nature.com/protocolexchange/protocols/349). The Barnes Maze with video tracking system was purchased from San Diego Instruments (San Diego, Calif.). ANY-maze video tracking software (Stoelting Co., Wood Dale, Ill.) was used for behavioral analysis. Briefly, the maze consists of 20 exploration holes with only one hole leading to a recessed escape box during task acquisition, on an elevated platform (
21 DIV rat hippocampal neurons transfected with eGFP-tagged exogenous αS species were suspended, stained for viability, and analyzed via flow cytometry (
CHIR-99021 and FK506 were purchased from Sigma Aldrich (St. Louis, Mo.). Both drugs were prepared as stock solutions (CHIR-99021: 5 mM and FK506 1 mM) in fresh DMSO and stored at −20° C. in aliquots. Either drug or DMSO vehicle were applied to cultured cells on DIV 16 with appropriate dilutions, five days prior to imaging or electrophysiology experiments.
All statistics were performed in Prism 6 (Graphpad Software, San Diego, Calif.) or Origin (OriginLab, Northampton, Mass.) software. Except where discussed above, one- and two-way ANOVA were used for univariate and two-variable analysis respectively. If ANOVA revealed significant variance between all groups, post-hoc analysis was performed using Bonferroni analysis adjusted for multiple groups. Univariate cumulative frequency distributions were compared using the unmodified Kolmogorov-Smirnov goodness of fit test. For all, statistical significance was set for α=0.05. Data representations are described in respective figure legends.
Although most PD cases are sporadic, familial PD can be caused by the duplication or triplication of the WT αS gene (SNCA) as well as point mutations, including the A53T or A30P mutation. Tg mouse lines expressing WT and mutant αS at various levels were used to test effects of these genetic mutations on synaptic responses. Western blots were used to determine the expression levels of both mouse and human αS in four mouse lines: 12-2 mice expressing WT human αS, H5 mice expressing A53T human αS at a lower level, G2-3 mice expressing A53T human αS at a higher level, and O2 mice expressing A30P human αS (
Next, whole-cell patch-clamp recordings of CA1 pyramidal neurons were performed in acute hippocampal slices from 3- to 6-month-old mice from each line (
Synaptic plasticity such as long-term potentiation (LTP) is known to increase the synaptic recruitment of AMPA receptors to dendritic spines. Therefore, the results in
The effects observed in acute hippocampal slices could result from differences in neural circuit development rather than neuron-autonomous differences in postsynaptic responses. Thus, glutamatergic mEPSCs were recorded in cultured hippocampal neurons from TgNg, 12-2, H5, G2-3, and O2 mice (
In neuronal cultures established from Tg mice (
The above results show that only the A53T mutation caused postsynaptic deficits even though both A30P and E46K mutations are linked to autosomal dominant PD. Unlike other kindred with familial PD, one unique pathological feature of PD brains from Contursi kindred, who carry the A53T mutation, is the frequent concurrence of both αS and tau pathology (Duda et al. 2002 Acta Neuropathol. 104(1):7-11). Tau missorting to dendritic spines is associated with memory loss and postsynaptic AMPA receptor signaling in FTDP-17 and Alzheimer's disease. Thus, it was tested whether there was a mechanistic relationship between A53T αS, tau, and postsynaptic deficits. Hippocampal neurons from H5 and G2-3 mice, which express A53T αS, were cultured, and their TgNg littermates were used as the control. These neurons were co-transfected with DsRed and three eGFP-tagged tau constructs (
Tau missorting is known to cause functional deficits in dendritic spines, which also depend upon tau phosphorylation. Therefore, it was also tested whether A53T αS-induced synaptic dysfunction is mediated by tau phosphorylation (
To further clarify the postsynaptic roles of αS, whether pharmacological blockade of αS-initiated tau mislocalization can rescue deficits in AMPA receptor signaling was tested (
Mislocalization of phospho-tau to dendritic spines leads to reduced mEPSC amplitude by reducing the surface levels of AMPARs. To determine if this reduction in surface levels of AMPARs also occurs with αS-dependent postsynaptic deficits, low density cultures of hippocampal neurons from G2-3 and TgNg mouse lines were treated with CHIR or vehicle and the live neurons were stained with a FITC-conjugated antibody against the N-terminus of GluA1 subunits (N-GluA1; Liao et al. 1999 Nature Neuroscience 2(1):37-43). Next, the neurons were fixed and permeabilized and stained with an antibody against PSD-95 to reveal the location of dendritic spines (see representative images in
Given that A53T αS causes a loss of surface GluA1, calcineurin-mediated AMPAR internalization was hypothesized to play a role in synaptic deficits caused by A53T αS. Calcineurin is a Ca2+-dependent protein phosphatase that mediates AMPAR internalization under multiple conditions including LTD, morphine treatment, neuronal toxicity, and exposure to Aβ. Calcineurin involvement was examined by recording mEPSCs from neurons expressing A53T αS in the presence of the calcineurin inhibitor FK506 (tacrolimus) or the vehicle (
While hyperexpression of WT, A30P, and A53T human αS results in presynaptic deficits, the results of this Example demonstrate that A53T αS causes unique additional deficits in postsynaptic neuronal function (
This Example also characterizes a postsynaptic signaling cascade that directly links the A53T αS mutation to tau-dependent pathophysiology (
Consistent with a previous report by Paumier et al. (2013 PlosOne 8(8):e70274), w aged G2-3 mice were found to exhibit deficits in synaptic plasticity and spatial memory tests. However, the studies reported herein extend the previous analysis by describing a novel molecular basis for synaptic deficits caused by A53T αS (that is, tau-missorting and postsynaptic deficits). Tau missorting to dendritic spines has been shown to be associated with cognitive deficits in models of Alzheimer's disease, FTDP-17, and stress. These results suggest that A53T mutation-induced tau missorting may contribute to dementia observed in the Contursi kindred. However, A53T-linked familial PD is not uniquely associated with dementia; dementia is seen in humans with SNCA multiplications and E46K mutations as well. It is possible that αS abnormalities can cause dementia via multiple mechanisms, which would be consistent with the well-documented clinical heterogeneity of PD. That is, although this study illuminates one possible pathway connecting tau pathology to familial A53T αS PD, it may also be relevant to other synucleinopathies with concurrent tauopathic dementia.
As shown in
Expected outcomes: The AP peptide is expected to block tau missorting to dendritic spines, rescue synaptic deficits, and ameliorate memory deficits, providing direct in vivo evidence that validates the usage of the peptide to treat Alzheimer's disease.
To test the in vivo efficacy of AP peptide for treating FTDP-17, AP peptide will be infused by an osmotic pump into the lateral ventricles of transgenic mice expressing P301L tau. The mouse model is described in Hoover et al. 2010 Neuron 68(6):1067-81. The perfusion method is described in Zhao et al. 2016 Nat. Med. 22(11): 1268-1276. Tau missorting to dendritic spines will be determined by biochemistry; memory deficits will be evaluated by the Morris water maze behavioral test and synaptic dysfunction will be assessed by electrophysiology.
Expected outcomes: The AP peptide is expected to block tau missorting to dendritic spines, rescue synaptic deficits, and ameliorate memory deficits, providing direct in vivo evidence that validates the usage of the peptide to treat FTDP-17.
To test the in vivo efficacy of AP peptide for treating Parkinson's disease, AP peptide will be infused by an osmotic pump into the lateral ventricles of transgenic mice expressing A53T α-synuclein. The mouse model is described in Teravskis et al. 2018 J. Neurosci. 38(45):9754-9767 and Lee et al., 2002 Proc Natl Acad Sci USA 99:8968-8973. The perfusion method is described in Zhao et al. 2016 Nat. Med. 22(11): 1268-1276. Tau missorting to dendritic spines will be determined by biochemistry; memory deficits will be evaluated by the Morris water maze behavioral test and synaptic dysfunction will be assessed by electrophysiology.
Expected outcomes: The AP peptide is expected to block tau missorting to dendritic spines, rescue synaptic deficits, and ameliorate memory deficits, providing direct in vivo evidence that validates the usage of the peptide to treat Parkinson's disease.
To test the in vivo efficacy of AP peptide for treating chronic traumatic encephalopathy (CTE), AP peptide will be infused by an osmotic pump into the lateral ventricles of a traumatic brain injury (TBI) model rat. In the TBI model, mechanical deformations of the rat brain are induced with high accuracy and very fast speed. The AP peptide will be infused to the lateral ventricles during the TBI surgery. One to two weeks after surgery, tau missorting to dendritic spines will be determined by biochemistry; memory deficits will be evaluated by the Morris water maze behavioral test; and synaptic dysfunction will be assessed by electrophysiology.
Expected outcomes: The AP peptide is expected to block tau missorting to dendritic spines, rescue synaptic deficits, and ameliorate memory deficits, providing direct in vivo evidence that validates the usage of the peptide to treat CTE.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/636,523, filed Feb. 28, 2018, which is incorporated by reference herein in its entirety.
This invention was made with government support under grants NS084007 and NS096437 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62636523 | Feb 2018 | US |
