Patent Application
20230034660
This disclosure concerns peptides that upregulate N-methyl-D-aspartate (NMDA) receptor activity by inhibiting binding of the GluN2A receptor subunit to zinc transporter 1 (ZnT1). This disclosure further concerns use of the peptides, such as for treating schizophrenia.
Zinc is a dynamic signaling element in the brain, critically contributing to sensory processing (Anderson et al., 2017; Patrick Wu and Dyck, 2018) and synaptic plasticity (Li et al., 2001a; Huang et al., 2008; Pan et al., 2011; Eom et al., 2019). The zinc transporter ZnT3 (Slc30a3) packages the metal into synaptic vesicles of large populations of excitatory neurons in the cerebral cortex, hippocampus, amygdala and dorsal cochlear nucleus, among other brain regions (Cole et al., 1999). Vesicular zinc is synaptically released front ZnT3-containing terminals in an activity-dependent manner (Assaf and Chung, 1984; Vogt et al., 2000), and similar to classical neurotransmitters, diffuses across the synaptic cleft (Anderson et al., 2015) to act on a variety of postsynaptic receptors (Ruiz et al., 2004; Besser et al., 2009; Kalappa et al., 2015; Perez-Rosello et al., 2015), including the N-methyl-D-aspartate (NMDA) receptor (NMDAR) (Peters et al., 1987; Jo et al., 2007; Vergnano et al., 2014; Anderson et al., 2015). As zinc is not metabolized, it is likely transported into cells or bound to protein complexes to terminate its synaptic function. The fact there are 24 different zinc transporters as well as a large number of zinc-binding proteins present in cells (Kambe et al., 2014), is indicative of the complex processes involved in regulating cellular zinc. However, little is known about how these metal regulatory systems influence synaptic zinc.
GluN2A-containing NMDA receptors are major targets of synaptically-released zinc due to their sensitivity to nanomolar concentrations of extracellular zinc, a negative allosteric modulator of receptor function (Paoletti et al., 1997; Rachline et al., 2005). It is generally assumed that synaptic release alone provides sufficient accumulation of zinc in the synaptic cleft to account for its inhibition of NMDARs (Vergnano et al., 2014). Indeed, this is perhaps the simplest explanation for zinc's synaptic action. However, this model only takes into account ZnT3's contribution to synaptic zinc, despite the complex, albeit poorly understood, transport system for the metal, indeed, ZnT3 is not the only zinc transporter located at or near the synapse. ZnT1 (Slc30a1), a cell membrane transporter that shuttles zinc from the cytoplasm to the extracellular space, not only localizes to the postsynaptic density (Qin et al., 2009; Shusterman et al., 2014; Sindreu et al., 2014), but also binds directly to the GluN2A subunit of NMDARs (Mellone et al., 2015). This positions ZnT1 to act as a postsynaptic regulator of synaptic zinc, in concert with ZnT3-dependant presynaptic release.
Schizophrenia is a complex and disabling psychological disorder that affects 1% of the world population. Symptoms of schizophrenia include hallucinations, delusions, and disordered thinking, speech and behavior. Studies in both humans and animal models have suggested that NMDA hypofunction plays a role in this disease (Snyder and Gao, Front Cell Neurosci 7:31, 2013; Lindsley et al., Curr Top Med Chem 6(8):771-785, 2006). Agents that enhance NMDA receptor function may serve as therapeutic agents for this disorder (Balu, Adv Pharmacol 76:351-382, 2016).
Peptides derived from subunit GluN2A of the human NMDA receptor that are capable of blocking binding of GluN2A to ZnT1 are described by the present disclosure. The peptides enhance NMDA receptor function and can be used, for example, in the treatment of conditions associated with NMDA receptor hypofunction, such as schizophrenia.
Provided herein are isolated or synthetic peptides derived from human GluN2A. In some embodiments, the peptides include at least six consecutive amino acid residues of SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 14, each of which represent a fragment of GluN2A that was shown herein to interact with ZnT1. In some embodiments, the peptide is no more than 20 amino acids in length and shares at least 90% sequence identity to human GluN2A, set forth herein as SEQ ID NO: 21. In some examples, the peptide is 9 to 15 amino acids in length. In some examples, the peptide includes at least one chemical modification or at least one non-natural amino acid, such as to enhance protease resistance.
Fusion proteins that include a GluN2A peptide disclosed herein and a heterologous protein are also provided. In some embodiments, the heterologous peptide is a cell-penetrating peptide.
Further provided are compositions that include a GluN2A peptide or fusion protein, and a pharmaceutically acceptable carrier.
Nucleic acid molecules and vectors including the GluN2A peptides and fusion proteins disclosed herein are further provided by the present disclosure.
Methods of inhibiting binding of GluN2A to ZnT1 in a cell, such as a neuronal cell, are also provided herein. In some embodiments, the method includes contacting the cell with a peptide, fusion protein, composition, nucleic acid molecule or vector disclosed herein. The method can be an in vitro method or an in vivo method.
Also provided are methods of treating schizophrenia by administering to a subject suffering from schizophrenia a therapeutically effective amount of a peptide, fusion protein, nucleic acid or vector disclosed herein.
The foregoing and other objects and features of the disclosure will become more apparent front the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Nov. 24, 2020, 17.8 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ NOs: 1-20 are amino acid sequences of GluN2A peptides.
SEQ ID NO: 21 is the amino acid sequence of human GluN2A,
SEQ ID NO: 22 is the amino acid sequence of a scrambled peptide.
SEQ ID NO: 23 is the amino acid sequence of the trans-activator of transcription (TAT) cell-penetrating peptide.
SEQ ID NOs: 24-27 are nucleic acid primer sequences.
AC auditory cortex
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AMPAR AMPA receptor
CAPS N-cyclohexyl-3-aminopropanesulfonic acid
CCal corticocallosal neurons
CCol corticollicular neurons
ChR2 channel rhodopsin
CMV cytomegalovirus
CV coefficient of variance
DCN dorsal cochlear nucleus
DRG dorsal root ganglia
eGFP enhanced green fluorescent protein
EPSC excitatory postsynaptic current
Fmoc 9-fluorenylmethoxy carbonyl
GABAAR γ-aminobutyric acid type A receptor
GluN2A NMDA receptor 2A
IC50 inhibitory concentration 50
KO knockout
MNI 4-Methoxy-7-nitroindolinyl
mOsm milliosmole
N2AZ GluN2A-ZnT1
NMDA N-methyl-D-aspartate
NMDAR NMDA receptor
NMDG N-methyl-D-glucamine
PLA proximity ligation assay
PN peripheral neuron
PPR paired pulse ratio
RCA rolling circle amplification
RT room temperature
scN2AZ scrambled control N2AZ
AT trans-activator of transcription
TTX tetrodotoxin
WT wild-type
ZnT1 zinc transporter 1
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.
As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to he understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:
Administer: As used herein, administering a composition (e.g. a peptide) to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, intraperitoneal, intravenous, intrathecal, topical, oral, subcutaneous, intramuscular, intranasal, intramuscular or by direct injection into a tissue.
Cell-penetrating peptide (CPP): Peptides that facilitate the cellular uptake of another protein or molecular caro linked by a covalent bond or non-covalent interaction. CPPs generally deliver cargo into a cell by endocytosis. In many instances, CPPs have an amino acid composition that is rich in charged amino acids, such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged. amino acids and non-polar/hydrophobic amino acids.
Contacting: Placement in direct physical association; includes both in solid and liquid form.
Effective amount (or therapeutically effective amount): The amount of an agent (such as a GluN2A peptide, fusion protein, nucleic acid or vector disclosed herein) that is sufficient to effect beneficial or desired results. A therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The beneficial therapeutic effect can include enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition. In one embodiment, an “effective amount” is an amount sufficient to reduce symptoms of a disease, disorder or condition, for example by at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (as compared to no administration of the therapeutic agent).
Fusion protein: A protein containing amino acid sequence from at least two different (heterologous) proteins or peptides. In some examples herein, the fusion protein comprises a portion of a GluN2A. protein and a cell-penetrating peptide. Fusion proteins can be generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons. Fusion proteins, particularly short fusion proteins, can also be generated by chemical synthesis.
GluN2A: A subunit of the heterotrimeric NMDA receptor. An exemplary amino acid sequence of human GluN2A is set forth herein as SEQ ID NO: 21. GluN2A is encoded by the GRIN2A gene.
Heterologous: A heterologous protein or polypeptide refers to a protein or polypeptide derived from a different source or species.
Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell, blood or tissue of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include those purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.
N-methyl-D-aspartate receptor (NMDAR): An ionotropic glutamate receptor and ion channel protein found in nerve cells. The receptor is a heterotrinieric complex comprised three different subunits: GluN1, GluN2 and GluN3. There are eight different isoforms of GluN1. due to alternative splicing. There are four different subunits of GluN2 (GluN2A, GluN2B, GluN2C and GluN2D) and two different subunits of GluN3 (GluN3A and GluN3B).
Non-natural amino acid: Non-proteinogenic amino acids, which amino acids that are not naturally encoded or found in the genetic code of any organism. Non-natural amino acids are also referred to as “unnatural amino acids.” Peptides that incorporate non-natural amino acids are often more stable and more resistant to proteases than their naturally occurring counterparts. Examples of non-natural amino acids include, for example, D-amino acids, homo-amino acids, β-homo amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids and α-methyl amino acids. D-amino acids are the mirror image, of the naturally occurring L-isomers. Homo-amino acids are amino acids with a methylene (CH2) group added to the α-carbon of an amino acid. A βeta-homo-amino acid is an analog of a standard amino acid in which the carbon skeleton has been lengthened by insertion of one carbon atom immediately after the acid group. An N-methyl amino acid possesses a methyl group at the nitrogen instead of a proton. An α-methyl amino acid has a methyl group substituted for the proton on the α-carbon atom of the amino acid.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in the methods disclosed herein are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing. Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of peptides.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween, sodium acetate or sorbitan monolaurate.
Polypeptide or peptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide,” “peptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The terms “polypeptide” and “peptide” are specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.
A conservative substitution in a polypeptide is a substitution of one amino acid residue in a protein sequence for a different ammo acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, a protein or peptide including one or more conservative substitutions (for example no more than 1, 2, 3, 4 or 5 substitutions) retains the structure and function of the wild-type protein or peptide. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be readily selected by testing antibody cross-reactivity or its ability to induce an immune response. Examples of conservative substitutions are shown below.
Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.
Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein.
Schizophrenia: A serious disabling mental disorder characterized by an abnormal interpretation of reality, hallucinations, delusions, disordered thinking and behavior, and disorganized speech
Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls in the Biosciences 8, 155-65, 1992; and Pearson et al,, Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mel. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment, methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals (including research subjects such as rodents). A subject is also referred to herein as a “patient.” In some embodiments herein, the subject has schizophrenia.
Synthetic: Produced by artificial means in a laboratory, for example a synthetic polypeptide can be chemically synthesized in a laboratory.
Therapeutically effective amount: A dose sufficient to prevent advancement of a disease, or to cause regression of the disease, or which is capable of reducing symptoms caused by the disease, such as cerebral ischemia.
Zinc transporter 1 (ZnT1): A protein that mediates zinc transport through cell membranes. In humans, ZnT1 is encoded by the SLC30A1 gene.
Described herein are peptides derived from the C-terminal region of human GluN2A, a subunit of the NMDA receptor. The disclosed peptides are capable of interfering with binding of GluN2A to ZnT1. Blocking binding of GluN2A with ZnT1 results in upregulation of the NMDA receptor function. Use of the peptides for treating disorders associated with NMDA hypofunction, such as schizophrenia, is also described.
Provided herein are isolated or synthetic GluN2A peptides comprising an amino acid sequence derived from one of three fragments of the C-terminal region of human GluN2A (set forth herein as SEQ ID NO: 21). In some embodiments, the peptide. comprises at least 6, at least 7, at least 8 or all 9 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 14, wherein the peptide is no more than 20 amino acids in length and shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to human GluN2A of SEQ ID NO: 21. In some embodiments, the peptide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length and comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or at least 14 consecutive amino acids of any one of SEQ NOs: 1-20. In specific examples, the peptide comprises or consists of any one of SEQ ID NOs: 1-20.
In some embodiments, the peptide includes at least one chemical modification, such as a modification to improve protease resistance and/or increase stability of the peptide. In some examples, the chemical modification is an N-terminal acetylation, a C-terminal amidation, or both.
In some embodiments, the peptide includes at least one non-natural amino acid, such as a non-natural amino acid that confers increased stability. In some embodiments, the at least one non-natural amino acid includes one or more of a D-amino acid, a homo-amino acid, a β-homo amino acid, a proline derivative, a pyruvic acid derivative, a 3-substituted alanine derivative, a glycine derivative, a ring-substituted phenylalanine derivative, a ring-substituted tyrosine derivative, a linear core amino acid, and an N-methyl amino acid.
In some embodiments, the peptide includes at least one amino acid substitution, such as a conservative substitution, relative to any one of SEQ ILS NOs: 1-20, such as one, two, three, four or five amino acid substitutions relative to any one of SEQ ID NOs: 1-20.
Also provided are fusion proteins that include a GluN2A-derived peptide described herein and a heterologous protein. In some embodiments, the heterologous peptide is a peptide that promotes cellular uptake of the fusion protein, such as a cell-penetrating peptide (CPP). In some examples, the CPP is the TAT peptide of SEQ ID NO: 23. In other examples, the CPP is a peptide rich in charged amino acids, such as lysine or arginine. In other examples, the CPP contains an alternating pattern of polar/charged amino acids and non-polar/hydrophobic amino acids. In particular non-limiting examples, the CPP comprises poly-arginine, such as 6, 7, 8, 9, 10, 11 or 12 arginine residues. In other non-limiting examples, the CPP comprises poly-lysine, such as 6, 7, 8, 9, 10, 11 or 12 lysine residues.
In other embodiments, the heterologous protein or peptide is a protein tag, such as an affinity tag (for example, chitin binding protein, maltose binding protein, glutathione-S-transferase or poly-His), an epitope tag (for example, V5, c-myc, HA or FLAG) or a fluorescent tag (e.g., GFP or another well-known fluorescent protein).
Further provided herein are compositions comprising the polypeptide or fusion protein disclosed herein and a pharmaceutically acceptable carrier.
Also provided are isolated nucleic acid molecules encoding the GluN2A-derived peptides or fusion proteins disclosed herein. In some embodiments, the isolated nucleic acid molecule is operably linked to a promoter, such as a heterologous promoter. Vectors comprising the nucleic acid molecules are also provided by the present disclosure. Compositions comprising a nucleic acid molecule or vector disclosed herein and a pharmaceutically acceptable carrier are further provided.
Further provided s a method of inhibiting binding of CiluN2A to zinc transporter 1 (ZnT1) in cells, such as neuronal cells. In some embodiments, the method includes contacting the cells with a peptide, fusion protein, nucleic acid or vector disclosed herein. In some examples, the method is an in vitro method. In other examples, the method is an in vivo method comprising administering the peptide, fusion protein, nucleic acid or vector to a subject. In specific examples, the subject suffers from schizophrenia.
Methods of treating schizophrenia in a subject are further provided. In some embodiments, the method includes administering to a subject suffering from (or likely to suffer from) schizophrenia a therapeutically effective amount of a peptide, fusion protein, nucleic acid or vector disclosed herein.
The amino acid sequence of the GluN2A subunit of the human NMDA receptor is provided below. As described in Example 2, 61 human GluN2A-derived peptides, each 15 amino acids in length and overlapping by 14 amino acids, were synthesized and tested for their ability to inhibit binding of GluN2A to ZnT1. Three regions in the C-terminal portion of GluN2A that were shown to be involved in binding to Zn1 were identified; these regions arc indicated in bold underline in the human GluN2A sequence below.
tprvlnscsn
rrvykkmpsi esdv
As shown in
Based on Peptides 2-8:
NDSYLRSSL
NDSYLRSSLRSTASY
Based on Peptides 40-42:
ANKNNMYST
YAANKNNMYSTPR
YAANKNNMYSTPRVL
Based on Peptides 48-52:
PRVLNSCSN
TPRVLNSCSNR
TPRVLNSCSNRRVYK
Provided herein are isolated or synthetic GluN2A peptides capable of disrupting binding of GluN2A to ZnT1. In some embodiments, the GluN2A peptide is 6 to 20 amino acids in length, and the amino acid sequence of the peptide comprises at least 6 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 14. In some embodiments, the peptide is 9 to 15 amino acids in length, and the amino acid sequence of the peptide comprises at least 9 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 14. In some examples, the peptide shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with human GluN2A of SEQ ID NO: 21. In specific non-limiting examples, the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 1-20.
Also contemplated are variants of the GluN2A peptides, such as variants exhibiting increased stability and/or increased affinity for ZnT1. Thus, in some embodiments, provided are GluN2A peptides comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 1-20, or a portion thereof (such as a portion about 6, about 7, about 8, about 9, about 11, about 13, about 15 or about 20 amino acids in length). In other embodiments, provided are GluN2A polypeptides comprising no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 amino acid substitution(s), such as conservative substitutions. In some embodiments, the GluN2A peptides include at least one chemical modification, such as N-terminal acetylation and/or C-terminal amidation, and/or at least one non-natural amino acid.
CPPs are a family of polypeptides that facilitate transduction of proteins, nucleic acids or other compounds across membranes a receptor-independent manner (Wadia and Dowdy, Curr. Protein Pept. Sci. 4(2):97-104, 2003). Typically, CPPs are short polycationic sequences that can facilitate cellular uptake of compounds to which they are linked into endosomes of cells.
The capacity of certain peptides to deliver proteins or nucleic acids into cells was originally described for the HIV-encoded Tat protein, which was shown to cross membranes and initiate transcription. It was then discovered that the portion of the Tat protein that was required for the transduction of the protein was only an 11 amino acid polypeptide, referred to as the Tat peptide (YGRKKRRQRRR; SEQ ID NO: 23). When fused with other proteins, the Tat peptide has been demonstrated to deliver these proteins, varying in size from 15 to 120 kDa, into cells in tissue culture (Frankel and Pabo, Cell 55(6):1189-93, 1988; Green and Loewenstein, J. Gen. Microbiol. 134(3):849-55, 1988; Vives et al., J. Biol. Chem. 2721″25):16010-7, 1997; Yoon et al., J. Microbiol. 42(4):328-35, 2004; Cai et al., Eur. Pharm. Sci. 27(4):311-9, 2006).
Other known CPPs include PENETRATIN™, a 16 amino acid peptide derived from the third helix of the Drosophila Antennapedia homeobox gene (U.S. Pat. No. 5,888,762; Derossi et al., J. Biol. Chem. 269:10444-10450, 1994; Schwarze et al., Trends Pharmacol. Sci. 21:45-48, 2000); transportan, a 27 amino acid chimeric peptide comprised of 12 amino acids from the N-terminus of the neuropeptide galanin and the 14-amino acid protein mastoparan, connected via a lysine (U.S. Pat. No. 6,821,948; Pooga, FASEB J. 12:67-77, 1998; Hawiger, Curr. Opin. Chem. Biol. 3:89-94, 1999); peptides front theVP22 protein of herpes simplex virus (HSV) type 1 (Elliott et al., Cell 88:223-233, 1997); the UL-56 protein of HSV-2 (U.S. Patent Application Publication No. 2006/0099677); and the Vpr protein of HIV-1 (U.S. Patent Application Publication No. 2005/0287648). In addition, a number of artificial peptides also are known to function as CPPs, such as poly-arginine, poly-lysine and others (see, for example, U.S. Application Publication Nos. 2006/0106197; 2006/0024331; 2005/0287648; and 2003/01.25242; Zhibao et al., Mol. Ther. 2:339- 347, 2000; and Laus et al., Nature Biotechnol. 18:1269-1272, 2000).
In some examples, the CPP is the TAT peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23.
In some examples, the CPP is rich in charged amino acids, such as lysine or arginine. In other examples, the CPP contains an alternating pattern of polar/charged amino acids and non-polar/hydrodrophobic amino acids. In particular non-limiting examples, the CPP comprises poly-arginine, such as 6, 7, 8, 9, 10, 11 or 12 arginine residues. In other non-limiting examples, the CPP comprises poly-lysine, such as 6, 7, 8, 9, 10, 11 or 12 lysine residues.
Methods of administering therapeutic proteins and peptides are well known in the art. In some embodiments of the disclosed methods, GluN2A peptides and fusion proteins are administered to a subject for the treatment of schizophrenia. When administering GluN2A peptides (or fusion proteins thereof), one must consider the appropriate target site based on the disease to be treated. If the site of action is the central nervous system, the protein must be able to cross the blood brain barrier (BBB) or be delivered directly to the target site in the brain.
Methods of administering neurotrophic factors for the treatment of a variety of neurodegenerative diseases has been previously described (see, for example, Levy et al., Biodrugs 19(21:97-127, 2005; Gill et al., Nat Med 9:589-595, 2003; Nutt et al., Neurology 60:69-73, 2003; Olson et al., J Neural Transm Park Dis Dement Sect 4:79-95, 1992; Eriksdotter et al., Dement Geriatr Cogn Disord 9:246-257, 1998; Bradley, Ann Neurol 38:971, 1995; The BDNF Study Group Phase III, Neurology 52:1427-1433, 1999; Ochs et al., Amyotroph Lateral Scler Other Motor Neuron Disord 1:201-206, 2000; ALS CATS Treatment Study Group, Neurology 46(5):1244-1249, 1996; Miller et al., Neurology 47:1329-1331, 1996; Miller et al., Ann Neurol 39:256-260, 1996: Lai et al., Neurology 49:1621-1630, 1997; Borasio et al., Neurology 51:583-586, 1998).
In some embodiments, the GluN2A peptide or fusion protein is administered intraperitoneally, such as by intraperitoneal injection.
In some embodiments, the GluN2A peptide or fusion protein is administered by direct infusion into the brain, such as by intracerebroventricular (ICV) injection/infusion, intrastriatal injection, intranigral injection, intracerebral injection, infusion into the putamen, intrathecal infusion (such as by using an implanted pump) or by subcutaneous injection. Intranasal administration of peptides also leads to delivery to the CNS. Thus, in some examples, the GluN2A peptide or fusion protein is administered intranasally.
In some embodiments, GluN2A peptides or fusion proteins are administered using biodegradable microparticles (˜1-100 μm) or nanoparticles (˜50-1000 nm). Nanoparticles and microparticles (also known as nanospheres or microspheres) are drug delivery vehicles that can carry encapsulated drugs such as synthetic small molecules, proteins, peptides, cells and nucleic acid based biotherapeutics for either rapid or controlled release. A variety of molecules (e.g., proteins, peptides and nucleic acid molecules) can be efficiently encapsulated in nanoimicroparticles using processes well known in the art.
The nano/microparticles for use with the methods described herein can be any type of biocompatible particle, such as biodegradable particles, such as polymeric particles, including, but not limited to polyarnide, polycarbonate, polyalkene, polyvinyl ethers, and cellulose ether nano/microparticles. In some embodiments, the particles are made of biocompatible and biodegradable materials. In some embodiments, the particles include, but are not limited to particles comprising poly(lactic acid) or poly(glycolic acid), or both poly(lactic acid) and poly(glycolic acid). In particular embodiments, the particles are poly(D,L-lactic-co-glycolic acid) (PLGA) particles.
Other biodegradable polymeric materials are contemplated for use with the methods described herein, such as poly(lactic acid) (PLA) and polyglycolide (PGA). Additional useful nano/microparticles include biodegradable poly(alkylcyanoacrylate) particles (Vauthier et al., Adv. Drug Del. Rev. 55: 519-48, 2003).
Various types of biodegradable and biocompatible nano/microparticles, methods of making such particles, including PLGA particles, and methods of encapsulating a variety of synthetic compounds, proteins and nucleic acids, has been well described in the art (see, for example, U.S. Publication No. 2007/0148074; U.S. Publication No. 20070092575: U.S. Patent Publication No. 2006/0246139: U.S. Pat. Nos. 5,753,234; 7,081,489: and PCT Publication No. WO/2006/052285). In addition, microsphere-mediated delivery of proteins to the central and peripheral nervous system has been described in, for example, U.S. 2011/0217264.
Embodiment 1. An isolated or synthetic peptide comprising at least 6 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 14, wherein the peptide is no more than 20 amino acids i- length and shares at least 90% sequence identity to human GluN2A of SEQ ID NO: 21.
Embodiment 2. The isolated or synthetic peptide of Embodiment 1, wherein the peptide is 9 to 15 amino acids in length.
Embodiment 3. The isolated or synthetic peptide of Embodiment 1 or Embodiment 2, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 1-8.
Embodiment 4. The isolated or synthetic peptide of Embodiment 1 or Embodiment 2, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 9-13.
Embodiment 5. The isolated or synthetic peptide of Embodiment 1 or Embodiment 2, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 14-20.
Embodiment 6. The isolated or synthetic peptide of any one of Embodiments 1-5, wherein the peptide comprises at least one chemical modification or non-natural amino acid.
Embodiment 7. The isolated or synthetic peptide of Embodiment 6, wherein the at least one chemical modification comprises an N-terminal acetylation, a C-terminal amidation, or both.
Embodiment 8. The isolated or synthetic peptide of Embodiment 6, wherein the at least one non-natural amino acid comprises a D-amino acid, a homo-amino acid, a β-homo amino acid, a proline derivative, a pyruvic acid derivative, a 3-substituted alanine derivative, a glycine derivative, a ring-substituted phenylalanine derivative, a ring-substituted tyrosine derivative, a linear core amino acid, or an N-methyl amino acid.
Embodiment 9. A fusion protein comprising the isolated or synthetic peptide of any one of Embodiments 1-8 and a heterologous protein.
Embodiment 10. The fusion protein of Embodiment 9, wherein the heterologous protein comprises a cell-penetrating peptide.
Embodiment 11. The fusion protein of Embodiment 10, wherein the cell-penetrating peptide comprises the amino acid sequence of SEQ ID NO: 23.
Embodiment 12. A composition comprising the peptide or fusion protein of any one of Embodiments 1-11 and a pharmaceutically acceptable carrier.
Embodiment 13. An isolated nucleic acid molecule encoding the peptide or fusion protein of any one of Embodiments 1-11.
Embodiment 14. The isolated nucleic acid molecule of Embodiment 13, operably linked to a heterologous promoter.
Embodiment 15. A vector comprising the isolated nucleic acid molecule of Embodiment 13 or Embodiment 14.
Embodiment 16. A method of inhibiting binding of GluN2A to zinc transporter 1 (ZnT1) in neuronal cells, comprising contacting the neuronal cells with the peptide, fusion protein, nucleic acid or vector of any one Embodiments 1-15.
Embodiment 17. The method of Embodiment 16, wherein the method is an in vitro method.
Embodiment 18. The method of Embodiment 16, wherein the method is an in vivo method comprising administering the peptide, fusion protein, nucleic acid or vector to a subject.
Embodiment 19. The method of Embodiment 18, wherein the subject suffers from schizophrenia.
Embodiment 20. A method of treating schizophrenia, comprising administering, to a subject suffering from schizophrenia a therapeutically effective amount of the peptide, fusion protein, nucleic acid or vector of any one Embodiments 1-15.
Embodiment 21. Use of the peptide, fusion protein, nucleic acid or vector of any one Embodiments 1-15 in the preparation of a medicament for the treatment of schizophrenia,
Embodiment 22. The peptide, fusion protein, nucleic acid or vector of one Embodiments 1-15 for use in a method of treating schizophrenia.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
The Examples describe examination of the role of ZnT1/GluN2A binding to synaptic zinc inhibition of NMDARs using a cell-permeant peptide selectively interfering with the interaction between the two proteins. Using this peptide, the critical contribution of ZnT1 to the actions of synaptic zinc was resolved, revealing a complex, circuitous route the metal must take in order to inhibit NMDAR function following its release from presynaptic terminals.
This example describes the experimental methods and materials used for the studies described in Example 2.
Cortical cultures were prepared from embryonic day 16 rats as previously described (McCord et al., 2014) (Hartnett et al., 1997). Briefly, pregnant rats (Charles River Laboratory) were sacrificed via CO2 inhalation. Embryonic cortices were dissociated with trypsin and plated at 670,000 cells per well on glass coverslips in six-well plates. Non-neuronal cell proliferation was inhibited after 2 weeks in culture with cytosine arabinoside (1-2 μM). Cultures were utilized at 3-4 weeks in vitro for PLA and electrophysiology experiments.
Human embryonic kidney tSA201 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1% GlutaMAX, as previously described (Glasgow and Johnson, 2014). Cells were plated in 35 mm petri dishes with three 15 mm glass coverslips treated with poly D-lysine (0.1 mg/ml) and rat-tail collagen (0.1 mg/ml) at a density of 1×105 cells/dish. Eighteen to 30 hours after plating, the cells were co-transfected using FuGENE 6 Transfection Reagent with cDNA encoding enhanced green fluorescent protein (eGFP), for identification of transfected cells, and WT rat NMDAR subunits GluN1-1a (GluN1; GenBank X63255) and GluN2A (GenBank M91561 in pcDNA1). GluN1-1 a and eGFP were expressed using a specialized pCl-neo vector with cDNA encoding eGFP inserted between the CMV promoter and the GluN1 open reading frame (Yi et al., 2018). At the time of transfection, 200 μM d1-APV was added to culture medium to prevent NMDAR-mediated cell death (Boeckman and Aizenman, 1996). For experiments testing the effect of N2AZ and scN2AZ, cells were incubated with 3 μM peptide for 3-6 hours prior to recording.
Proximity ligation assays were performed using Duolink PLA kit. Cortical cultures (3-4 weeks in vitro) were treated overnight with either N2AZ or scN2AZ (3 μM, dissolved in water). Coverslips were fixed in ice cold methanol for 5 minutes, rinsed in phosphate buffered saline (PBS) then permeabilized with 0.1% Triton-X in PBS. Coverslips were then incubated with primary antibodies, including rabbit anti-ZnT1, mouse anti-GluN2A, and chicken anti-Map2 antibodies. Coverslips were incubated with a donkey anti-chicken fluorescent secondary antibody to visualize neuron morphology. The PLA reaction was then completed according to DuoLink PLA protocol. Briefly, coverslips were incubated in DuoLink secondary antibodies (rabbit, mouse) conjugated with PLA oligonucleotides. Ligation solution was added to hybridize the PLA probes, allowing the oligonucleotides to join in a closed loop when secondary antibodies were in close proximity. Next the reaction was amplified with rolling-circle amplification (RCA) using the closed loop hybridized probes as a template. PLA probes were fluorescently labeled with oligonucleotides which hybridized to the RCA product during amplification. Coverslips from sister cultures were treated with either scN2AZ or N2AZ and reactions were run at the same time using the same preparation of reagents. Coverslips were mounted on glass slides using DuoLink mounting media and 4 random fields of view were imaged from each coverslip using a 60× oil objective on a Nikon A1R laser scanning confocal. PLA puncta were counted automatically using Fiji ImageJ (Version 2.0) software using maximum intensity projection of 8 sequential images in the z plane. All images were normalized to the same intensity threshold using the Yen threshold setting prior to automated quantification of punctae.
Male and female mice (postpartum days 18-28) were anesthetized with isoflurane and sacrificed. Brains were rapidly dissected and sectioned into 210 μm thick coronal slices containing dorsal cochlear nucleus (DCN) on a vibratome (Leica, VT1000S). Slices were incubated in ACSF containing 130 mM NaCl, 3 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 20 mM NaHCO3, 3 mM HEPES, and 10 mM glucose, saturated with 95% O2/5% CO2 (vol/vol), pH ˜7.3, ˜300 mOsm at 35° C. for 1 hour before being moved to room temperature. During preparation, ACSF was treated with Chelex 100 resin to remove any contaminating zinc. After applying Chelex to the ACSF, high-purity calcium and magnesium salts were added (99.995% purity). All plastic and glassware were washed with 5% high-purity nitric acid.
Whole-cell voltage-clamp recordings from tSA201 cells were performed 18-30 hours after transfection. Pipettes were fabricated from borosilicate capillary tubing (OD=1.5 mm, ID=0.86) using a Flaming Brown P-97 electrode puller (Sutter Instruments) and fire-polished to a resistance of 2.5-4.5 MΩ with an in-house fabricated microforge. Intracelluiar pipette solutions consisted of 130 mM CsCl, 10 mM HEPES, 10 mM BAPTA, and 4 mM MgATP with pH balanced to 7.2±0.05 using CsOH and final osmolality of 280±10 mOsm. Extracellular recording solution contained 140 mM NaCl, 2.8 mM KCl, 1 mM CaCl2, 10 mM HEPES, 10 mM tricine, and 0.1 mM glycine and was balanced to pH 7.2±0.05 and osmolality 290±10 mOsm with NaOH and sucrose, respectively. Glutamate (Glu), and ZnCI2 were diluted from concentrated stock solutions in extracellular solution each day of experiments. Buffered Zn2+ solutions were prepared via serial dilution, as previously described (Paoletti et al., 1997) (Serraz et al., 2016). Extracellular solutions were delivered to the cell using an in-house fabricated fast perfusion system (Glasgow and Johnson, 2014). Whole-cell currents were recorded using an Axopatch 200A patch-clamp amplifier (Molecular Devices), low-pass filtered at 5 kHz, and sampled at 20 kHz in pClamp10.7 (Molecular Devices). In all recordings from tSA201 cells, series resistance was compensated 85-90% and an empirically determined −6 mV liquid junction potential between the intracellular pipette solution and the extracellular recording solution was corrected.
The effect of the N2AZ on Zn2 + inhibition of GluN1/2A receptors was determined using the protocol shown in
where IZn/IGlu was calculated as the mean current over the final 1 second of Zn2+ application divided by the average of the mean steady state currents (final 1 second) elicited by Glu before and after Zn2+ application. A (IZn/IGlu at saturating Zn2+), IC50 and nH (Hill coefficient) were free parameters during fitting. Curve fitting and statistical comparisons were performed in Prism 8. IC50s were compared by one-way ANOVA.
Whole-cell recordings from cultured cortical neurons were obtained with glass micropipettes (3-6 MΩ) containing 140 mM CsF, 10 mM CsEGTA, 1 mM CaCl2, 10 mM HEPES, pH=7,2, 295 mOsm. Recording solution contained 150 mM NaCl, 2.8 mM KCl, 1.0 m CaCl2, 10 mM HEPES, 10 mM μM glycine, pH=˜7.2 , ˜300 mOsm. Using ephus (Suter et al. 2010) and a Multiclamp 700B amplifier (Molecular Devices), NMDAR excitatory postsynaptic currents (EPSCs) were recorded in voltage clamp (held at −70 mV) in the presence of TTX (300 nM to prevent synaptic activity), DNQX (20 μM, AMPA and kainate receptor antagonist), and 4-Methoxy-7-nitroindolinyl (MNI)-caged glutamate (40 μM). Neurons were visualized by including 10 μM Alexa 594 in the internal solution. To evoke NMDAR EPSCs, MNI-caged glutamate was photolytically unaged onto dendrites 120 μm from the cell soma using 1 ms pulses of UV-laser light (355 nm, DPSS Lasers). The ZX1-mediated potentiation for each cell was calculated as the average percent increase in responses following application of the metal chelator across these 4 uncaging locations.
For brain slice recordings, whole-cell recordings of NMDAR EPSCs in slice DCN cartwheel cells were obtained with micropipettes (3-6 MΩ) containing 128 mM Cs(CH3O3S), 4 mM MgCl2·6H2O, 4 mM Na2ATP, 10 mM HEPES, 0.3 mM Tris-GTP, 10 mM Tris-phosphocreatine, 1 mM CsEGTA, 1 mM, QX-314, 3 mM sodium ascorbate, pH=˜7.2, 300 mOsm. Cartwheel cells were identified by the presence of complex spikes (Zhang and Oertel, 1993; Golding and Oertel, 1997; Tzounopoulos et al., 2004) in cell-attached configuration before break-in or in response to current injections in current-clamp mode after break-in. NMDAR EPSCs were recorded in voltage clamp mode, at a holding potential of +40 mV, in the presence of DNQX (20 μM, AMPA and kainate receptor antagonist), SR95531. (20 μM, GABAAR antagonist), and strychnine (1 μM, GlyR antagonist), ZX1 (100 μM) was included in the pipette in experiments where noted. Whole-cell recordings of AMPAR EPSCs were obtained with micropipettes containing 113 K-gluconate, 4.5 mM MgCl2·6 H2O, 14 mM Tris-phosphocreatine, 9 mM HEPES, 0.1 mM EGTA, 4 mM Na2ATP, 0.3 mM Tris-GTP, 10 mM sucrose, pH=7.3, 295 mOsm. AMPA EPSCs were recorded in voltage clamp mode at a holding potential of −70 mV in the presence of SR9551. (20 μM, GABAAR antagonist) and strychnine (1 μM, GlyR antagonist). Both NMDAR and ANWAR EPSCs were evoked using an Isoflex stimulator (A.M.P.I. 0.1 ms pulses) stimulating parallel fibers with voltage pulses through a theta glass electrode. Stimulus intensity was adjusted to a level that consistently evoked stable responses. For paired pulse experiments, inter-stimulus interval was 50 milliseconds. Once a stable response was established, ZX1 (100 μM) was added to the recording solution to measure the effect of zinc chelation on EPSCs. The series resistance was not compensated because the currents measured were relatively small, therefore there was minimum voltage clamp error. The series resistance was monitored during the recording by delivering −5 mV voltage steps for 50 milliseconds for each sweep. The peak current value (ΔIpeak) generated immediately after the step in the command potential was used to calculate series resistance (Rseries) using the following formula: Rseries=−5 mv/ΔIpeak. The difference between baseline and steady-state current (ΔIss) was used to calculate input resistance (RI) using the following formula: RI=−5 mV/ΔI−Rseries. Recordings were excluded from further analysis if the series resistance or membrane resistance changed by more than 20% compared to the baseline period. Data were low-pass-filtered at 4 kHz and sampled at 10 kHz. NMDAR EPSC peak values were averaged over a 20-millisecond time window using custom Matlab 2012a software. All values reported are animal-based values, in cases where multiple cells were recorded from the same animal preparation, the average of cells is presented (Lazic et al., 2018). All recordings were performed at room temperature.
Quantitative Real-Time PCR (qPCR)
For qPCR analysis of rat cortical cultures, cells were harvested at 5, 12, 19 and 26 days in vitro and RNA was isolated using Invitrogen PureLink RNA Mini Kit. cDNA was synthesized from RNA transcripts using iScript Select cDNA Synthesis kit using Eppendorf Thermocycler. qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix. Relative expression was calculated using β-actin as a reference gene. Custom primers were designed using NCBI Primer-BLAST:
HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing: 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mm glutamine, and 10% (v/v) fetal calf serum in a 5% CO2 humidified atmosphere at 37° C. To express ZnT1, HEK 293 cells were transfected with ZnT1 or empty plasmid (control) using CaPO4 precipitation. Briefly,1 μg mouse ZnT-1 (pCMV6:ZnT1 Gen Bank Q60738) or empty vector plasmid (pCMV6, Origene) was incubated with 2 M calcium chloride in HEPES buffered solution containing 1.5 Na2HPO4 to generate a co-precipitate, this solution was then dispersed onto cultured cells for 6 hours. Twenty four hours later, cells were treated with the scN2AZ or N2AZ (3 μM ) overnight. To visualize intracellular zinc, cells were loaded with the fluorescent zinc indicator FluoZin-3 (2 μM) for 25 minutes at room temperature before imaging. Cells were imaged using 480 nm excitation filter and an emission 525 nm long pass filter on a Zeiss Axiovert 100 inverted microscope with a Polychrome IV monochromator (T.I.L.L. Photonics) and a cooled CCD camera (PCO). To measure zinc efflux, cells were superfused with Ringer's solution (120 mM NaCl, 0.8 mM MgCl, KCl, 5.4 mM CaCl 1.8, 20 mM HEPES, 15 mM glucose) and 1 μM Zn2+ with 5 μM pyrithione were added for 150 seconds. The FluoZin-3 signal was normalized to an initial baseline of 10 seconds in each experiment. Rates of initial decrease of the fluorescent signal following exposure to Zn2+ were determined during a 100 second period. For each experiment, at least 30 cells were imaged per coverslip and rates were averaged for 3-5 coverslips performed at 3 independent experiments. Fluorescence imaging measurements were acquired using Axon Imaging Workbench 5.2 (INDEC BioSystems) and analyzed using Excel and Prism GraphPad.
Far-Western protein-binding affinity assays were performed as previously described (Brittain et al., 2011; Yeh et al., 2017) (Moutal et al., 2017). Peptide spot arrays (15 mers) spanning the proximal C-terminus residues 1390-1464 of mouse GluN2A (Uniprot# P35436) in overlapping 1 aa steps were constructed using the Spots-synthesis method. Standard 9-fluorenylmethoxy carbonyl (Fmoc) chemistry was used to synthesize the peptides and spot them onto nitrocellulose membranes pre-derivatized with a polyethylene glycerol spacer (Intavis). Fmoc protected and activated amino acids were spotted in 20-30 arrays on 150 by 100 mm membranes using an Intavis MultiPep robot, The nitrocellulose membrane containing the immobilized peptides was soaked in N-cyclohexyl-3-aminopropariesulfonic acid (CAPS) buffer (10 mM CAPS, pH 11.0, with 20% v/v methanol) for 30 minutes, washed once with Tris-buffered 0.1% Tween 20 (TBST), and then blocked for 1 hour at room temperature (RT) with gentle shaking in TBST containing 5% (w/v) nonfat milk and then incubated with enriched Flag-tagged ZnT1 (SLC30a1) protein overnight at 4° C. with gentle shaking. Next, the membrane was incubated in primary antibody for Flag for 1 hour at RT with gentle shaking, followed by washing with TBST. Finally, the membrane was incubated in secondary antibody for 45 minutes, washed 3 times for 5 minutes in TBST, and visualized by infrared fluorescence (Li-Cor). Four independent peptide spot arrays were used in this study. A second set of membranes (n=4) was treated as above, but also in the presence of 100 μM of either N2AZ or scN2AZ and compared to 0.1% DMSO. For each experiment, an additional peptide array was done with omission of Flag-tagged ZnT1 (SLC30a1) protein to measure and correct for the background due to the primary and secondary antibodies.
Slice electrophysiology experiments using N2AZ and scN2AZ were completed blind to the identity of the peptide. Experiments in ZnT3 knockout and wild type animals were completed blind to the genotype. Electrophysiology recordings in cortical cultures and DCN slices were obtained using Ephus software run in Matlab 2012a (Mathworks). Cell parameters and response peaks were calculated using custom Matlab scripts. For neuronal culture electrophysiology, ZX1 potentiation was measured as the percent increase in NMDAR amplitude 5 minutes after the application of ZX1. In slice experiments, ZX1 potentiation was calculated as the average percent increase over baseline of NMDAR or AMPAR EPSCs 10-15 minutes after the addition of ZX1 (
This example describes peptides that disrupt binding of ZnT1 to GluN2A, resulting in enhancement of NMDA receptor function.
To study the effect of ZnT1 on NMDAR modulation, a peptide was designed to disrupt the ZnT1-GluN2A interaction. First, a peptide spot array was constructed, which spanned 74 amino acids of the C-terminal domain (residues 1390-1464) of mouse GluN2A (Uniprot# P35436), previously shown to be necessary for GluN2A-ZnT1 binding (Mellone et al., 2015). The array consisted of 61 15-mers (Table 1), each sequentially overlapping by 14 amino acids, similar to previously described procedures (Brittain et al., 2011; Yeh et al., 2017). Next, the peptide spot arrays were probed with flag-tagged ZnT1-enriched cell lysates and ZnT1 binding was visualized and quantified with immunofluorescence against the flag tag (
Next, rat neuronal cortical cultures were utilized to determine whether N2AZ treatment was sufficient to disrupt GluN2A-ZnT1 binding in vitro. First, however, ZnT1 expression in cortical cultures was verified using quantitative PCR. It was observed that ZnT1 mRNA expression increased developmentally over the first four weeks in vitro (
Zinc inhibits GluN2A-containing NMDARs through its high-affinity binding site on the extracellular, N-terminal domain of the GluN2A subunit (Paoletti et al., 1997; Vergnano et al., 2014; Anderson et al., 2015). Since ZnT1 shuttles neuronal intracellular zinc to the extracellular space, it was hypothesized that ZnT1 functionally localizes zinc in close proximity to its binding site on GluN2A and thereby contributes to the inhibition of NMDARs by the metal. To test this hypothesis, cortical cultures were treated with N2AZ or scN2AZ (3 μM) overnight prior to recordings of NMDAR-receptor mediated electrophysiological responses. NMDAR-mediated currents were evoked by photolytic uncaging of MNI-caged glutamate (40 μM) along the dendrite of a neuron under whole cell voltage clamp (
In order to investigate whether ZnT1 contributes to synaptic zinc-mediated inhibition of NMDAR, electrophysiological recordings were performed in acutely-prepared brain stem slice containing the dorsal cochlear nucleus (DCN), containing zinc-rich synaptic terminals emanating from parallel fibers. Synaptic zinc is released in this preparation in an activity-dependent manner, producing a pronounced inhibition of both NMDA and AMPA-mediated synaptic currents in cartwheel cells, the most prominent interneuron in the DCN. Synaptic NMDAR-mediated currents were isolated by voltage-clamping cartwheel cells at +40 mV to relieve the Mg2+ block, While recording in the presence of DNQX (20 μM). Slices were treated with either scN2AZ or N2AZ (3 μM) for at least 1 hour prior to ZX1 (100 μM) application. Parallel fibers were stimulated at 20 Hz, a frequency where zinc inhibition of NMDAR is entirely ZnT3-dependent (Anderson et al., 2015). It was found that N2AZ significantly reduced ZX1 potentiation of NMDAR-mediated synaptic currents, when compared to scN2AZ (
Stimulation of parallel fibers at high frequencies (100 Hz-150 Hz) uncovers a ZnT3-independent component of zinc inhibition of NMDAR EPSCs (Anderson et al., 2015). The contribution of the ZnT1-GluN2A interaction to this inhibition was next evaluated. It was found that N2AZ treatment eliminated ZX1 potentiation of NMDAR EPSCs at 100 Hz, stimulation frequency, suggesting that ZnT1-GluN2A interaction is required for ZnT3-independent zinc inhibition (
In addition to blocking NMDAR, synaptic zinc can inhibit AMPAR EPSCs in cartwheel cells (Kalappa et al., 2015). To test whether the aforementioned actions of N2AZ are specific for NMDAR ESPCs, the effect of N2AZ and scN2AZ on zinc inhibition of AMPAR EPSCs was measured. ZX1 potentiated ANWAR EPSCs to a similer extent regardless of the treatment (
Next, it was evaluated whether a change in presynaptic release of glutamate could contribute to the observed actions of N2AZ on NMDAR-mediated synaptic currents. Two independent measures of release probability were used, paired pulse ratio (PPR) and coefficient of variance (CV) of AMPAR responses. PPR inversely correlates with probability of release, while CV is the ratio of the standard deviation of EPSCs over the mean and it varies inversely with probability of release. To measure PPR, two stimuli were given in rapid succession and the ratio of the second EPSC to the first was calculated. It was found N2AZ and scN2AZ altered neither PPR nor CV (
Next, it was examined whether N2AZ could modify ZnT1-dependent zinc transport. Following an intracellular zinc load, decreases in intracellular zinc levels were measured over time as a readout of zinc transport in HEK293 cells previously transfected with a plasmid encoding for ZnT1 or an empty vector. FluoZin-3 fluorescence was utilized to quantify intracellular zinc levels (Qin et al., 2009; Shusterman et al., 2014). Cells were briefly treated with zinc pyrithione (1 μM Zn2+, 5 μM sodium pyrithione) to increase intracellular zinc concentrations until reaching a maximum steady-state level (
To control for potential effects of N2AZ on NMDAR affinity for zinc itself, the extent of inhibition by exogenous application of the metal onto tSA201 cells previously transfected with plasmids encoding GluN1 and GluN2A was measured (Glasgow and Johnson, 2014; Glasgow et al., 2017). Zinc was applied across a wide range of concentrations (1-300 μM) using a multi-barreled rapid-perfusion system while recording a 1 mM glutamate-evoked steady-state current. The calculated IC50's for zinc block in vehicle, N2AZ, or scN2AZ treated cells were essentially identical across the three treatments (IC50(nM); Vehicle: 21.82±2.09, n=5; scN2AZ: 23.37±2.43, n=5; N2AZ=25.64±2.22 n=5, Ordinary one-way ANOVA, p=0.4996), indicating that N2AZ does not affect NMDAR's affinity for the metal. Taken all of these results together, it was concluded that N2AZ reduces zinc inhibition of NMDARs by disrupting the GluN2A-ZnT1 interaction, without affecting glutamate release, ZnT1-dependent zinc transport, or zinc affinity for GluN1/2A receptors.
The simplest model to explain the results thus far is that the GluN2A-ZnT1 interaction is necessary for zinc inhibition, presumably by transporting zinc from the cytoplasm of the postsynaptic cell to the extracellular space, in close proximity to the NMDAR. This model predicts that intracellular zinc is needed for synaptic zinc inhibition of NMDARs. To test this hypothesis, intracellular zinc was chelated with ZX1 and the effects of extracellular ZX1 on NMDAR EPSCs was measured. ZX1 (100 μM) was included in the recording pipette, allowing the solution to diffuse into the patched cell for at least 30 minutes prior to applying extracellular ZX1 to assess zinc inhibition of NMDAR EPSCs. It was observed that intracellular ZX1 blocked extracellular ZX1 potentiation of NMDAR ESPCs (
It was next examined whether the well-established routes of entry for zinc in neurons, including calcium-permeable AMPAR (Weiss et al., 1993), and L-type calcium channels (Kerchner et al., 2000; Park et al., 2015), mediate potential translocation of synaptic zinc into cartwheel cells. AMPA receptors were immediately ruled out by the fact that all experiments were performed in the presence of DNQX. As prior reports have shown that L-type calcium channels can also bind to ZnT1 (Levy et al., 2009; Shusterman et al., 2017), it was examined whether these channels contribute to synaptic zinc inhibition of NMDARs. Nimodipine, 20 μM was used for at least 20 minutes prior to recordings, to inhibit L-type calcium channels and zinc inhibition of NMDAR responses was measured. No significant differences were observed in ZX1 potentiation of NMDAR EPSCs between nimodipine-treated slices (23.6±6.9%, n=4) and vehicle-treated (DMSO) slices (23.6±9.4% n=6, unpaired t-test, p=0.997) (
Current models suggest that zinc inhibition of synaptic NMDARs depends exclusively on presynaptically-released zinc. In contrast, the results disclosed herein indicate that zinc inhibition of NMDAR EPSCs also requires postsynaptic zinc and the presence of GluN2A-ZnT1 association. The results demonstrate that the physical dissociation of GluN2A and ZnT1 by the newly developed peptide N2AZ diminished the inhibitory actions of synaptic zinc on NMDAR EPSCs. Moreover, chelation of postsynaptic zinc abolished zinc inhibition of NMDARs. Prior to the work presented here, it had been generally assumed that zinc cleft concentrations following its synaptic release are sufficient to directly inhibit NMDAR function (Vogt et al., 2000; Pan et al., 2011; Vergnano et al., 2014; Anderson et al., 2015; McAllister and Dyck, 2017), without involvement of the transport pathway uncovered by the present disclosure. However, it is shown herein that ZinT1-GluN2A association is necessary for zinc to be rapidly localized to physiologically relevant microdomains in very close proximity to the GluN2A-containing NMDARs. Indeed, this is highly reminiscent of calcium microdomains that have been postulated for a number of synaptic functions, including rapid synaptic release of neurotransmitters (Llinas et al., 1992.; Blackstone and Sheng, 2002; Berridge, 2006; Stanley, 2016). Whether similar transport processes are in place for synaptic zinc to activate or modify other known postsynaptic targets for the metal, including the metabotropic zinc receptor GPR39 (Besser et al., 2009) or AMPAR-mediated synaptic currents (Kalappa et al., 2015), remains to be determined.
Why is such an indirect signaling path necessary for synaptic zinc inhibition of NMDARs?This may be the result of the complex nature of zinc as a signaling molecule itself (Kay and Toth, 2008; Paoletti et al., 2009; Pan et al., 2011). As alluded to earlier, zinc is a promiscuous ligand that acts on a variety of postsynaptic targets (Hershfinkel et al., 2001; Ruiz et al., 2004; Perez-Rosello et al., 2013; Kalappa et al., 2015; Perez-Rosello et al., 2015). Moreover, not all vesicles at zinc-rich synaptic terminals contain zinc (Wenzel et al., 1997; Lavoie et al., 2011), and zinc-containing vesicle release probability can change with varying levels of activity (Quinta-Ferreira and Matias, 2005; Lavoie et al., 2011). Therefore, maintaining adequate signaling requires precise spatial zinc regulation, in addition to presynaptic release. The interaction between ZnT1 and GluN2A may be reflective of a system that harnesses and direct zinc's signaling properties, while supplying and maintaining specificity of action for a given activity level. As NMDAR function is regulated by subunit composition (Cull-Candy and Leszkiewicz, 2004; Yashiro and Philpot, 2008; Paoletti et al., 2013), as well as by its localization in postsynaptic structures (Parsons and Raymond, 2014), ZnT1 may endow the zinc-containing synapse with a dynamic form of regulation specific for GluN2A-containing NMDAR signals.
ZnT1 expression is also tightly coupled to fluctuations in free intracellular zinc levels (Nishito and Kambe, 2019). Rises in intracellular zinc concentrations are quickly detected by the metal regulatory element (MRE) transcription factor 1 (MTF1) (Zhao et al., 2014) to induce upregulation of MRE-driven genes, including ZnT1 (Hardyman et al., 2016). As increases in intracellular zinc levels have been prominently detected following neuronal depolitrization (Li et al., 2001 b; Sheline et al., 2002), it is also conceivable that the ZnT1-GluN2A complex is a key component of activity-dependent synaptic processes, perhaps even in synapses that do not express ZnT3, and thereby, vesicular zinc. In fact, robust NMDAR activation can lead to intracellular zinc liberation from metal binding proteins such as metallothionein (Aizenman et al., 2000) independent of synaptic zinc (Sensi et al., 1997; Vander Jagt, et al., 2009), likely as a consequence of glutamate-stimulated production of oxygen and nitrogen-derived reactive species (Dawson et al., 1991; Lafon-Cazal et al., 1993; Reynolds and Hastings, 1995). The observed actions of N2AZ of ZnT 3-independent zinc inhibition of NMDAR-mediated responses (i.e. caged glutamate responses in cortical neurons in culture and 100 Hz stimulation of parallel fibers,
In summary, the present disclosure describes a cell-permeant peptide that dissociates the zinc transporter ZnT1 from the highly zinc sensitive NMDAR subunit GluN2A. This tool allowed for determination of the mechanism via which zinc inhibits NMDAR function, which involves not only extracellular ZnT3-dependent zinc but also intracellular zinc and ZnT1-GluN2A complexes. It is proposed that the ZnT1-GluN2A association allows the synapse to direct zinc to its high affinity binding site within the GluN2A-containing NMDAR by creating a physiologically- and spatially-distinct extracellular zinc microdomain in the synapse.
ICR mice were administered stereotaxic injections of retrograde microspheres of different colors to label CCal PNs and CCol neurons. CCol neurons were used to identify AC, and viral vector (AAV) for expression of ChR2 in AC L2/3 PNs (
In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.
This application claims the benefit of U.S. Provisional Application No. 62/942,979, filed Dec. 3, 2019, which is herein incorporated by reference in its entirety.
This invention was made with government support under grant numbers NS043277 and DC007905 awarded by the National Institutes of Health, and grant number 1655480 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/063016 | 12/3/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62942979 | Dec 2019 | US |
