Patent Application
20250051391
A computer readable form of the Sequence Listing “2223-P65207PC00” (81, 020 bytes) created on Dec. 15, 2022, is herein incorporated by reference.
The present disclosure relates to peptide inhibitors that reduce a-synuclein toxicity through binding with CHMPB2 and/or inhibiting the interaction between CHMPB2 and a-synuclein and their use in treating synucleopathies.
Protein-protein interactions (PPIs) govern virtually all molecular pathways involved in cell growth, differentiation, and survival (1, 2). Inhibition of PPIs with peptides or small molecules to modulate these pathways has proven to be successful for the treatment of cancers (3, 4). PPI inhibitors could conceivably be a promising new therapeutic venue for neurodegenerative proteinopathies, such as Parkinson's disease (PD), for which no disease-modifying therapies exist (5). Wild-type (WT) or mutant a-synuclein protein (a-syn) accumulates in PD to form oligomers that disrupt core cellular systems causing neurodegeneration (6). Rescuing these toxic effects by targeting PPIs has been an unexplored therapeutic strategy for PD (7, 8). Identification of a-syn sequence based putative PPI inhibitors that reduce a-syn accumulation have been described (9, 59).
A first aspect is directed to a method of decreasing a-syn levels and/or decreasing a-syn toxicity in a cell, the method comprising contacting the cell with a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor.
Another aspect is directed to a method of inhibiting neural degeneration, the method comprising administering to a subject in need thereof a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor.
Another aspect is directed to a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor comprising a peptide comprising i) a MIT-Interacting Motif (MIM) sequence or a sequence with at least 50% sequence identity to said MIM sequence; or 2) an a-syn interaction sequence or a sequence with at least 50% sequence identity to said a-syn interaction sequence, wherein the peptide inhibits CHMP2B-a-syn interaction by at least 50%.
A further aspect is directed to a peptide, the peptide consisting of 5 to 213 amino acids, preferably, 5 to 25 amino acids, 5 to 30 amino acids, or 7 to 30 amino acids, and comprising i) a MIT-Interacting Motif (MIM) sequence or a sequence with at least 50% sequence identity to said MIM sequence; or 2) an a-syn interaction sequence or a sequence with at least 50% sequence identity to said a-syn interaction sequence, wherein the peptide inhibits CHMP2B-a-syn interaction by at least 50%.
Yet another aspect is directed to a nucleic acid molecule comprising a polynucleotide sequence encoding any peptide or polypeptide described herein.
Another aspect is directed to a vector comprising a vector backbone and any nucleic acid molecule described herein.
A further aspect is directed to a recombinant cell recombinantly expressing any peptide or polypeptide, any nucleic acid molecule or any vector described herein.
Another aspect is directed to a composition comprising any peptide, polypeptide, CHMP2B:a-syn inhibitor, nucleic acid, vector or recombinant cell described herein.
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
An embodiment of the present disclosure will now be described in relation to the drawings in which:
Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term “a cell” includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art (see, e.g. Green and Sambrook, 2012).
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, (such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
The terms “about”, “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.
The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”. For ranges described herein, subranges are also contemplated, for example every, 0.1 increment there between. For example, if the range is 80% to about 90%, also contemplated are 80.1% to about 90%, 80% to about 89.9%, 80.1% to about 89.9% and the like.
The term “cell” as used herein refers to a single cell or a plurality of cells.
A “conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.
As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to any chain of two or more natural or unnatural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). The polypeptides incorporated into the biphasic vesicles of the disclosure can include for example from 3 to 3500 natural or unnatural amino acid residues. Included are proteins that are a single polypeptide chain and multisubunit proteins (e.g. composed of 2 or more polypeptides).
The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
The terms “nucleic acid” or “oligonucleotide” as used herein means two or more covalently linked nucleotides. Unless the context clearly indicates otherwise, the term generally includes, but is not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which may be single-stranded (ss) or double stranded (ds). For example, the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term “oligonucleotide” as used herein generally refers to nucleic acids up to 200 base pairs in length and may be single-stranded or double-stranded. The sequences provided herein may be DNA sequences or RNA sequences, however it is to be understood that the provided sequences encompass both DNA and RNA, as well as the complementary RNA and DNA sequences, unless the context clearly indicates otherwise. For example, the sequence 5′-GAATCC-3′, is understood to include 5′-GAAUCC-3′, 5′-GGATTC-3′, and 5′GGAUUC-3′.
The term “MIT-Interacting Motif (MIM) sequence” as used herein means a sequence that has been identified as such including those in
The term “a-syn interaction sequence” as used herein means an a-syn sequence comprising amino acids 103-114 (Accession number NM_000345.4) or a fragment thereof that is at least 5 amino acids long or at least 7 amino acids long (for example amino acids 103-109, 104-110, 105-111, 106-112, 107-1113 or 108-114, 103-110, 104-111, 105-112 . . . 103-111, 104 to 112 etc.) and that inhibit CHMP2B a-syn interaction by at least 50% (which can be referred to as biologically active fragments). The sequences can be mammalian such as human, and may be wildtype as for example as provided in. Decreased interaction can be assessed by comparing interaction in the presence or absence of the a-syn sequence or fragment, or by comparing interaction in the presence of the a-syn sequence or fragment compared to a control peptide such as a scrambled peptide.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Accumulation of a-syn into toxic oligomers mediates dopaminergic neurodegeneration in Parkinson's disease and other synucleinopathies. A high-throughput, proteome-wide screen was performed as described in the Examples which identified protein-protein interaction inhibitors that reduced a-syn oligomer formation and rescued a-synuclein toxicity. One of the peptide inhibitors disrupted a previously unknown interaction between the C-terminal region of a-synuclein and charged multivesicular body protein 2B (CHMP2B), a component of the endosomal sorting complex required for transport III (ESCRT-III). It is shown that, through this interaction, a-synuclein disrupts lysosomal activity thereby inhibiting its own degradation; this effect may be amplified upon a-syn oligomerization. Conversely, the peptide inhibitor restored lysosomal activity and thus led to decrease of a-syn levels, rescuing a-syn toxicity. It also lowered a-syn levels in preclinical rodent models of Parkinson's disease and in human cells harboring disease-causing a-synuclein mutations, including iPSC-derived dopaminergic neurons. Furthermore, the peptide inhibitor protected dopaminergic neurons from a-syn-mediated degeneration in C. elegans and preclinical rodent models of Parkinson's disease.
Accordingly a first aspect relates to a method of decreasing a-syn levels and/or decreasing a-syn toxicity in a cell, the method comprising contacting the cell with a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor.
As demonstrated herein, peptide inhibitors were identified that decreased a-syn levels and decreased a-syn toxicity in vitro and in vivo. As demonstrated in the Examples, a peptide comprising the amino acid sequence IPIQLKA (SEQ ID NO: 1) was identified as inhibitory in the screen and corresponds to residues 203-209 in uniprot ID: Q9UQN3. The inhibitory peptide identified in the screen mapped to the C-terminal domain of charged multivesicular body protein 2B (CHMP2B) MIT binding motif (MIM). The MIM has the sequence of EEIERQLKALG (SEQ ID NO: 2) and corresponds to residues 201-211 in uniprot ID: Q9UQN3.
Additional peptide inhibitors were identified and tested.
For example, different peptides based or related to the identified sequence or a MIM domain, for example from other CHMP proteins, were also able to inhibit interaction with a-syn or CHMP self interaction by at least 50%.
Accordingly in an embodiment, the CHMP2B:a-syn inhibitor is or comprises a peptide.
In an embodiment, the peptide is or comprises a CHMP2B MIM sequence.
The a-syn interaction domain with CHMP2B was also mapped.
In another embodiment, the peptide is or comprises an a-syn interaction sequence.
Various peptides can be used based on, for example, known MIM and a-syn sequences, variations thereof and sequences described herein. In an embodiment, the peptide is one that inhibits interaction of CHMP2B and a-syn or CHMP2B self interaction by at least 50%, at least 60%, at least 70% or at least 80%.
Inhibition of the interaction can be assessed for example by a competition assay, for example as described in the Examples. In some embodiments, the inhibition of the interaction can be assessed for example by a pull down assay (e.g. immunoprecipitation assay), optionally pulldown assays in cell culture (immunoprecipitating fora-syn and blotting for CHMP e.g. CHMP2B (or the other way around), in presence and absence of the peptide, or performing fluorescence polarization measurements of the CHMP2B/a-syn interaction in presence and absence of the peptide. The assays can also be compared to a control peptide such as a scrambled sequence peptide.
The peptide may be a naturally occurring sequence or a mutant such as a known mutant or other mutant that maintains the inhibitory activity of the peptide regarding CHMP2B a-syn interaction.
For example, there are known mutations in the CHMP2B MIT-binding motif (MIM), such as Q206H mutant. In addition, various MIM sequence variations were tested.
In an embodiment, the peptide comprises a wildtype MIM sequence.
In an embodiment, the peptide comprises a wildtype a-syn sequence.
In other embodiments, the peptide is a non-naturally occurring peptide for example ones demonstrated herein.
In an embodiment, the CHMP2B: a-syn inhibitor comprises a peptide comprising a MIT-Interacting Motif (MIM) sequence or a sequence with at least 50% sequence identity to said MIM sequence and that inhibits CHMP2B-a-syn interaction by at least 50%. In an embodiment, the peptide has a MIM sequence described in Table 1,
In an embodiment, the peptide comprises or comprises up to 1, 2, 3, 4 or 5 mutations (e.g. changes)/10 amino acids relative to a naturally occurring MIM sequence. Peptides, whether or not comprising mutations, that inhibit a-syn and CHMP2B interaction by at least 50%, at least 60%, at least 70% or at least 80% are contemplated.
In an embodiment, the peptide consists of a sequence of 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 7 to 30, 7 to 25, 7 to 20, 7 to 15 or 7 to 10 amino acids. The peptide can for example be 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, or 5 amino acids. In some embodiments, the peptide is at least 7 amino acids. In other embodiments, the peptide is 12 amino acids. In yet other embodiments, the peptide is 9 amino acids
The peptide can be of any length between 5 and for example 30 amino acids.
The peptide can comprise at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to said MIM sequence or said a-syn interaction sequence.
Where the peptide comprises additional CHMP or a-syn sequence (e.g. extending N and/or C terminal from said MIM or a-syn interaction sequence), the percent sequence identity can be at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to said CHMP or a-syn sequence.
For example, the peptide can be or comprise a C-terminal CHMP sequence comprising a MIM region, optionally a CHMP sequence described herein or one with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to such sequences. Preferably the CHMP sequence is a human CHMP sequence. For example, the peptide can comprise at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to a human CHMP C-terminal sequence.
A full length CHMP sequence, optionally CHMP2B sequence can also be used or fragments thereof comprising at least 5, at least 6, at least 7 or more amino acids of the MIM domain. In some embodiments, the CHMP fragment comprises between 5 to 213 amino acids, optionally 199 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 7 to 30 amino acids, 7 to 25 amino acids, 7 to 20 amino acids, 7 to 15 amino acids, or 12 amino acids. In some embodiments, the CHMP2B fragment is amino acid residues 1-199 in uniprot ID: Q9UQN3.
A-syn sequences up to 60 amino acids can also be used or fragments thereof comprising at least 5, at least 6, at least 7 or more amino acids of the a-syn interaction sequence. In some embodiments, the a-syn interaction sequence fragment comprises between 5 to 60 amino acids, 5 to 50 amino acids, 5 to 40 amino acids 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 7 to 30 amino acids, 7 to 25 amino acids, 7 to 20 amino acids, 7 to 15 amino acids, or 9 amino acids.
In some embodiments, the length of the peptide is any length between 5 and 213 amino acids long, optionally between 5 and 25 amino acids long, optionally between 5 and 50 amino acids long, optionally between 5 and 75 amino acids long, optionally between 5 and 100 amino acids long, optionally between 5 and 125 amino acids long, optionally, between 5 and 150 amino acids long, optionally between 5 and 200 amino acids long.
In some embodiments, the length of the peptide is any length between 7 and 213 amino acids long, optionally between 7 and 25 amino acids long, optionally between 7 and 50 amino acids long, optionally between 7 and 75 amino acids long, optionally between 7 and 100 amino acids long, optionally between 7 and 125 amino acids long, optionally, between 7 and 150 amino acids long, optionally between 7 and 200 amino acids long.
In an embodiment, wherein the peptide is at least 7 amino acids and/or less than 20 amino acids. In one embodiment, the peptide is 12 amino acids.
In an embodiment, the peptide has a positive overall charge.
In an embodiment, the peptide comprises IPIQLKA (SEQ ID NO: 1), or a sequence with at least 50% sequence identity to IPIQLKA (SEQ ID NO: 1) that inhibits CHMP2B-a-syn interaction, optionally at least 60%, at least 70% or at least 80% or at least 90% sequence identity to IPIQLKA (SEQ ID NO: 1) and that inhibits CHMP2B-a-syn interaction. In some embodiments the peptide comprises IPIQLKA (SEQ ID NO: 1), or a sequence with at least about 50%, optionally about 55%, optionally about 60%, optionally about 65%, optionally about 70%, optionally about 75%, optionally about 80%, optionally about 85%, optionally about 90%, sequence identity to IPIQLKA (SEQ ID NO: 1) and inhibits CHMP2B-a-syn interaction.
In an embodiment, the peptide comprises IERQLKA (SEQ ID NO: 16) (DPpep1.1), EIERQLKALG (SEQ ID NO: 17) (DPpep1.2), DEEIERQLKALG (SEQ ID NO: 6) (DPpep1.3), DEEIERQLDALG (SEQ ID NO: 18) (DPpep1.4), IPKQEKA (SEQ ID NO: 19) (DPpep1.5), EEDDDMKELENWAGSM (SEQ ID NO: 20) (CHMP4-MIM), DEELERRLKALK (SEQ ID NO: 21) (SUPER), EQDELSQRLARLRDQV (SEQ ID NO: 22) (1B-MIM), VPVKARPRQAELVAAS (SEQ ID NO: 23) (6-MIM2), EDQLSRRLAALR (SEQ ID NO: 3) (A1-MIM), DADLEERLKNLR (SEQ ID NO: 5) (2A-MIM), LEAMQSRLATLR (SEQ ID NO: 7) (3-MIM), FDDLSRRFEELK (SEQ ID NO: 13) (IST1-MIM), RNERQLKALG (SEQ ID NO: 24) (optim1), EEEIVRQLKALG (SEQ ID NO: 25) (optim 2), DIEIEFQLKALG (SEQ ID NO: 26) (optim 3), EIERQLKAQI (SEQ ID NO: 27) (optim 4), DEEYERQWKALG (SEQ ID NO: 28) (optim 5), DEAIERVLKALG (SEQ ID NO: 29) (optim 7) DDEIEVQLKALG (SEQ ID NO: 30) (optim.8), TLEIERQLKA (SEQ ID NO: 31) (optim9) or LEEIERQLKALG (SEQ ID NO: 32) (optim10).
In an embodiment, the peptide comprises a sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to IERQLKA (SEQ ID NO: 16) (DPpep1.1), EIERQLKALG (SEQ ID NO: 17) (DPpep1.2), DEEIERQLKALG (SEQ ID NO: 6) (DPpep1.3), DEEIERQLDALG (SEQ ID NO: 18) (DPpep1.4), IPKQEKA (SEQ ID NO: 19) (DPpep1.5), EEDDDMKELENWAGSM (SEQ ID NO: 20) (CHMP4-MIM), DEELERRLKALK (SEQ ID NO: 21) (SUPER), EQDELSQRLARLRDQV (SEQ ID NO: 22) (1B-MIM), VPVKARPRQAELVAAS (SEQ ID NO: 23) (6-MIM2), EDQLSRRLAALR (SEQ ID NO: 3) (A1-MIM), DADLEERLKNLR (SEQ ID NO: 5) (2A-MIM), LEAMQSRLATLR (SEQ ID NO: 7) (3-MIM), FDDLSRRFEELK (SEQ ID NO: 13) (IST1-MIM), RNERQLKALG (SEQ ID NO: 24) (optim1), EEEIVRQLKALG (SEQ ID NO: 25) (optim 2), DIEIEFQLKALG (SEQ ID NO: 26) (optim 3), EIERQLKAQI (SEQ ID NO: 27) (optim 4), DEEYERQWKALG (SEQ ID NO: 28) (optim 5), DEAIERVLKALG (SEQ ID NO: 29) (optim 7) DDEIEVQLKALG (SEQ ID NO: 30) (optim.8), TLEIERQLKA (SEQ ID NO: 31) (optim9) or LEEIERQLKALG (SEQ ID NO: 32) (optim10) and inhibits CHMP2B-a-syn interaction.
In some embodiments, the peptide comprises the amino acid sequence EEIERQLKALG (SEQ ID NO: 2). In some embodiments, the peptide comprises the amino acid sequence KEEEDDDMKELENWAGSM (SEQ ID NO: 33). In an embodiment, the peptide comprises a sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to EEIERQLKALG (SEQ ID NO: 2) or KEEEDDDMKELENWAGSM (SEQ ID NO: 33).
In an embodiment, the peptide comprises or is DEEIERQLKALG (SEQ ID NO: 6) (DPpep1.3).
In an embodiment, the peptide is or comprises amino acid residues 103-114 of a-syn (Accession number NM_000345.4) or a fragment thereof that is at least 5 amino acids long. In some embodiments, the peptide is or comprises amino acid residues 103-108, 103-109, 104-110, 104-113, 105-111, 105-112, 106-111, 106-112, 107-111, 107-113, 108-113 or 108-114 of a-syn (Accession number NM_000345.4). In some embodiments, the peptide has the amino acid sequence In an embodiment, the peptide is at least 5 amino acids of NEEGAPQEGILE (SEQ ID NO: 34), In an embodiment, the peptide is or comprises NEEGAPQEGILE (SEQ ID NO: 34) (corresponding to positions 103 to 114).
The peptide can comprise for example a sequence with at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% or greater identity to a MIM, CHMP, a-syn interaction sequence. Peptides that inhibit for example CHMP2B:a-syn interaction by at least 50% or greater as described herein are contemplated.
The peptides described herein may have 1, 2, 3 or 4 or more amino acid substitutions, optionally conservative substitutions. For example, a peptide with at least 50% sequence identity to a sequence of 7 amino acids in length, may have 1, 2, 3 or 4 substitutions, optionally conservative substitutions. The peptide may comprise for example up to 5 substitutions for every 10 amino acids. This can be calculated by looking at the full length of the peptide such that some stretches may have more than 5 substitutions/10 amino acids and others that are less than 5, with the overall number of substitutions not exceeding 5/10 amino acids.
Substitutions and mutations are used interchangeable.
The peptide may comprise one or more modifications, for example 2, 3, 4, 5, 6 or more modifications compared to a sequence described herein. In some embodiments, each amino acid of the peptide is modified. In some embodiments, the modification is stapling the peptide (for example, as shown for the cyclic bisphenyl conjugated peptides).
For example, the modification can be an amino acid modification, or a stability modification or both. As demonstrated in the results, the peptides can be modified to increase stability by stapling the peptides. Stapling the peptide can be achieved by covalently linking the sidechains of two amino acids using for example, a hydrocarbon moiety, a bishphenyl moiety, or an hexafluorobenzene, thereby forming a peptide macrocycle.
“Stapling” of a peptide refers to covalently linking two residues in a helical peptide (located such that they are on the same side of the helix, e.g., positions i (where i denotes the position of the first residue) and i+3 or positions i and i+7, using a linking moiety. This linking moiety can be a hydrocarbon moiety (hydrocarbon staple), or a different chemistry, such as a bisphenyl moiety. The modification can be carried out after the synthesis of the peptide. The stapling positions can for example be cysteine residues that are present or added to the peptide. After synthesis, the peptide can be chemically conjugated to the linking moiety, for example in the case of two cysteines being linked, the two cysteines are linked to the linking moiety (60).
In some embodiments the peptide has a positive charge. In some embodiments, the peptide lacks a C-terminal positive charge.
In an embodiment, the peptide is a stapled peptide. In some embodiments, the modification comprises stapling using for example a hydrocarbon or other stapling moiety.
Any of the peptides described herein can be stapled. In an embodiment, wherein the stapled peptide comprises a sequence selected from DEEYCRQWKALC (SEQ ID NO: 35), DEEICRQLDALC (SEQ ID NO: 37) or DIEICFQLKALC (SEQ ID NO: 36).
In some embodiments, the modification comprises inclusion of one or more non-canonical or D-amino acids. For example, the modification can comprise substituting one or more amino acids of the any of the peptides described herein with a D-amino acid or a non-canonical amin acid.
In some embodiments, the peptides are modified to increase stability, optionally wherein the modification comprises inclusion of non-canonical amino acids or generation of D-amino acids.
In some embodiments, the inhibitor, peptide or polypeptide is or comprises a peptide that reduces a-syn toxicity and/or CHMP self-interaction and/or CHMP2B/a-syn interaction. In some embodiments, the peptide inhibitor is any peptide that's reduces CHMP2B/a-syn interaction. In some embodiments, the inhibitor, peptide or polypeptide is or comprises any peptide that reduces a-syn toxicity and/or CHMP self-interaction and/or CHMP2B/a-syn interaction by at least about 50%, optionally about 55%, optionally about 60%, optionally about 65%, optionally about 70%, optionally about 75%, optionally about 80%, optionally about 85%, optionally about 90%, optionally about 95%, optionally about 96%, optionally about 97%, optionally about 98% or optionally about 99%.
As described herein the said peptide can comprise one or more exogenous residues, optionally interspersed within said peptide, for example for one or more, optionally two cysteine residues interspersed within said peptide, optionally wherein at least 4 amino acids (e.g. MIM sequence or a-syn sequence) is present between the exogenous residues. Exogenous residues are added for example for cyclizing a peptide. They may be interspersed within or added to an end or ends of a peptide. The peptide with or without exogenous residues is one that inhibits CHMP2B-a-syn interaction by at least 50% as described herein.
The inhibitor may comprise and/or the peptide may be conjugated to a transport moiety (optionally an amino acid sequence), for example a cell-penetrating moiety (such as tat peptide, cyclic cell penetrating peptide) and/or a blood-brain barrier penetrating moiety (such as an Angiopep peptide (Angiochemem; Montreal Candada) or blood brain crossing antibody such as an antibody or nanobody, optionally a transferrin antibody). Examples of engineered antibodies that can cross the blood brain barrier have been made for example as described in Ledford, H. Engineered antibodies cross blood-brain barrier. Nature (2011) herein incorporated by reference. Examples of cell penetrating peptides can be found in for example Xie, Jing et al. “Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application” Frontiers in pharmacology vol. 11 697. 20 May. 2020, which is hereby incorporated by reference. Examples of cyclic cell penetrating peptides can found in for example Qian, Ziqing et al. “Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides.” Biochemistry vol. 55,18 (2016): 2601-12, which is incorporated herein by reference. A review of strategies to deliver peptide drugs to the brain can be found at Laltsa, A et al Mol. Pharmaceutics 2014, 11, 4, 1081-1093, which is incorporated herein by reference.
In some embodiments, the peptide is conjugated to an aprotinin sequence or fragment thereof such as an Angiopep peptide. In one embodiment, the angiopep and the conjugated peptide has one of the following sequences: TFFYGGSRGKRNNFKTEEYDEEICRQLDALC (SEQ ID NO: 38) or TFFYGGSRGKRNNFKTEEYGEARCEIQDLLC (SEQ ID NO: 39) and is cyclic. In another embodiment, the angiopep and the conjugated peptide has one of the following sequences: TFFYGGSRGKRNNFKTEEYDEEIERQLDALG (SEQ ID NO: 40) or TFFYGGSRGKRNNFKTEEYGEARDEIQDLLE (SEQ ID NO: 41) and is non-cyclic. The peptide can be labelled, for example with FITC, for example for tracking.
Peptides can be synthesized or purchased from a custom peptide synthesis service available for example LifeTein (New Jersey). As described herein, the cyclic peptides were synthesized using an aminohexanoic acid spacer and for cyclic peptides creating a disulphide bridge between cysteine residues e.g. cyclic peptides can be synthesized as follows (N-Terminal: FITC-Ahx, C-Terminal: Amidation, Disulfide Bridge between Cysteine residues) and non-cyclic peptides can be synthesized as follows (N-Terminal: FITC-Ahx, C-Terminal: Amidation).
The cyclic peptides can be used as linear peptides. Alanine scan studies showed that modifications of these residues did not affect the activity of the peptides. Accordingly, the C residues for example used for cyclization can be replaced with other residues, for example alanine.
The inhibitor may also comprise and/or the peptide may be conjugated to other attachments. In some embodiments, the inhibitor comprises and/or the peptide is conjugated to a serum half-life extending moiety such as a lipid, Fc portion of an antibody or PEG by PEGylation.
The inhibitor may also comprise or consist of a polypeptide comprising the peptide and for example a cell penetrating peptide, a blood brain barrier penetrating peptide optionally a nanobody, or other type of peptide or antibody. The methods and uses disclosed herein can also comprise administering or use of the inhibitors, peptides and polypeptides described herein.
A further aspect is a polypeptide comprising a peptide described herein. For example, in an embodiment, the peptide consists of a 5 to 213 amino acids, preferably 5 to 30 amino acids. The peptide can comprise other lengths as described elsewhere.
Said peptide can comprise or consist of a MIT-Interacting Motif (MIM) sequence or a sequence with at least 50% sequence identity to said MIM sequence that inhibits CHMP2B-a-syn interaction by at least 50% as described herein. Said peptide can comprise or consist of a a-syn interaction sequence or a sequence with at least 50% sequence identity to said a-syn interaction sequence.
As also described herein the said peptide can comprise one or more exogenous residues, optionally interspersed for example for one or more, optionally two cysteines. Exogenous residues are added for example for cyclizing a peptide. They may be interspersed within or added to an end or ends of a peptide. The peptide with or without exogenous residues is one that inhibits CHMP2B-a-syn interaction by at least 50% as described herein.
The polypeptide can comprise any peptide described herein. In some embodiments, the polypeptide can comprise a TAT peptide or other cell-penetrating peptide, and/or blood brain barrier penetrating peptides such as angiopep, and/or a blood brain barrier crossing moiety such as a nanobody.
Similar to peptides described herein, the polypeptides can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Mode1396; Milligen/Biosearch 9600). Alternatively, the peptides, polypeptides, fragments or variants thereof described herein may be recombinantly produced using various expression systems as is well known in the art.
Also provided in another aspect is a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor comprising a peptide described herein.
The peptide can be any peptide described herein.
In some embodiments, the polypeptide or inhibitor comprises and/or the peptide is fused to a cell penetrating or blood-brain barrier penetrating moiety or to a serum half-life extending moiety such as a lipid, Fc portion of an antibody or PEG by PEGylation.
Another aspect is a nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide or peptide described herein. In an embodiment, the polynucleotide is codon optimized, for example for humans. The nucleic acid molecule can be used in methods described herein.
In some embodiments, the polynucleotide is delivered via any viral vector, optionally an AAV vector.
Also provided in another aspect is a vector comprising a vector backbone and a nucleic acid molecule described herein. For example, the vector backbone can be a lentivirus or an adeno associated virus.
The inhibitor can be a nucleic acid molecule or vector described herein.
A further aspect is a recombinant cell recombinantly expressing the polypeptide or peptide or comprising the nucleic acid molecule or vector described herein.
The cell targeted in the methods or the cells used to make the recombinant cell can be any cell. In some embodiments, the cells are neurons e.g. neurons can be targeted or the recombinant cell is a recombinant neural cell. In other embodiments, the cells are astrocytes, microglial cells, ependymal cells, or oligodendrocytes. In other embodiments, the cells are satellite cells or Schwann cells.
Recombinant cells can be made by for example being transformed, transfected or transduced with a vector comprising a nucleic acid, optionally any nucleic acid described herein. In some embodiments, the recombinant cells are HEK293T, HEK293S, HEK293F and/or CHO cells and may be used to produce recombinant peptides.
A further aspect is a composition comprising a peptide, a polypeptide, a CHMP2B:a-syn inhibitor, a nucleic acid molecule, a vector or a recombinant cell described herein.
The composition can also comprise a suitable diluent or carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.
Also provided are uses of the inhibitors, peptides, polypeptides, nucleic acid molecules, vectors, recombinant cells and compositions described herein as well as their use for manufacturing a medicament, for example for treating a synucleinopathy.
In an embodiment, the a-syn that is decreased is oligomerized a-syn. For example, the a-syn decreased may be oligomeric a-syn or the toxicity reduced may be due to a-syn. The a-syn can also be non-oligomerized a-syn.
The methods can be used where the cell is in vivo. For example the cell can be contacted by administering the by administering the CHMP2B:a-syn inhibitor to a subject in need thereof. The inhibitor is for a peptide inhibitor that inhibits interaction between CHMP2B and a-syn, for example decreasing the interaction in its presence by at least 50%.
The subject in need thereof can be a subject with a synucleinopathy.
For example, the synucleinopathy can be Parkinson's disease (PD). In some embodiments, the synucleinopathy is multiple system atrophy (MSA), dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer's Disease (AD), neurodegeneration with brain iron accumulation, Parkinson's disease dementia (PDD), Alzheimer's disease and/or prodromal PD/DLB/MSA (e.g., REM sleep behaviour disorder, primary autonomic failure, MCI-LB or DLB-MCI). In addition, there are biomarkers in development to identify people with alpha-synuclein aggregation ante-mortem (e.g., RT-QuIC assay using CSF and possibly PET imaging eventually). As such, in some embodiments, patients identified as having a-synuclein aggregation could benefit from therapies that reduce alpha-synuclein accumulation/aggregation such as inhibitors, peptides, polypeptides, nucleic acid molecules, vectors, recombinant cells and compositions described herein. There will also be the possibility of utility in prodromal conditions including but not limited to REM sleep behaviour disorder (RBD), primary autonomic failure and Gaucher disease.
The subject can comprise a disease causing mutation in a-syn gene. Mutations in a-syn gene have been associated with synucleinopathies. For example, the mutation can be one or more of the following: A30G, A30P, E46K, H50Q, G51D, A53E, A53T, A53V and/or SNCA multiplication (duplication, triplication) and/or a truncation mutation.
Also provided in an aspect is a method of inhibiting neural degeneration, the method comprising administering to a subject in need thereof a charged multivesicular body protein 2B: a-synuclein (CHMP2B:a-syn) inhibitor as described herein.
As demonstrated herein the inhibitor can decrease oligomerized a-syn and prevent neural degeneration.
In an embodiment, the subject in need thereof is a subject with a synucleinopathy such as Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer's Disease (AD), neurodegeneration with brain iron accumulation, Parkinson's disease dementia (PDD), Alzheimer's disease and/or prodromal PD/DLB/MSA (e.g., REM sleep behaviour disorder, primary autonomic failure, MCI-LB or DLB-MCI).
The inhibitors may be administered for example via IV administration, directly to the brain for example either transiently or with a permanent infusion catheter and pump, optionally the parenchyma and/or ventricle of the brain. In some embodiments, the inhibitor may be administered using gene therapy using for example nucleic acids and vectors described herein. In other embodiments, the inhibitor may be delivered via augmented delivery, optionally using focused ultrasound. In some embodiments, the inhibitor comprises a cell and blood-brain-barrier crossing moiety and is optionally administered intravenously. In some embodiments, the inhibitor is a stapled peptide which cross the cell membrane and may be injected into the brain directly or delivered using focused ultrasound. In some embodiments, inhibitor can be delivered using gene therapy vectors such as AAVs.
The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
The following non-limiting examples are illustrative of the present disclosure:
Proteomic Screen Identifies Peptides that Rescue a-Syn Cytotoxicity
A screening system to discover candidate peptides to perturb endogenous PPIs based on libraries of short linear interaction motifs that mediate a large fraction (˜30%) of human PPIs (10-12) was recently developed. Here, a lentiviral library of 50,549 7-mer peptide motifs (13) on a green fluorescent protein (GFP) scaffold (
To cause cytotoxicity relevant to PD, proteostatic stress in HEK293 cells was induced by inhibiting the proteasome with MG132 and inducing the overexpression of human a-syn (WT) from idiopathic PD in one screen and the more toxic A53T mutant from familial PD in a second screen. The combination of a-syn overexpression and pharmacological proteasome inhibition led to death of most cells, with only those that expressed a protective peptide surviving. By extracting genomic DNA from the surviving cells and amplifying the peptide coding sequences, the protective peptides (
To detect a-syn oligomers, a protein fragment complementation assay (PCA) of a-syn oligomerization in HEK293 cells (14) was used. In this assay, human A53T a-syn was fused to either the C- or N-terminal half of yellow fluorescent protein (YFP). No fluorescence is detectable while a-syn exists in monomers but, when a-syn oligomerizes, the two halves of YFP come in close enough proximity to form a functional, spontaneously fluorescent protein. This fluorescence has previously been shown to approximate levels of toxic oligomeric conformations of a-syn in cell and rodent models (15-17). By using fluorescence activated cell sorting (FACS), cells expressing Flag-tagged peptides that directly or indirectly interfered with a-syn oligomer formation were isolated and then the peptides were identified from genomic DNA (
The amino acid sequence of Pdpep1 was mapped to the C-terminal region of Charged Multivesicular Body Protein 2B (CHMP2B), a member of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery. This region corresponds to a MIT-Interacting Motif (MIM) (20) that is thought to mediate several protein-protein interactions. Using this mapping, optimization of Pdpep1 by generating several versions of the peptide of various lengths based on the relevant sequence of CHMP2B: IERQLKA (SEQ ID NO: 16) (Pdpep1.1), EIERQLKALG (SEQ ID NO: 17) (Pdpep1.2), and DEEIERQLKALG (SEQ ID NO: 6) (Pdpep1.3) was sought. It was found that Pdpep1.3 had the most prominent effects on cell survival under proteostatic stress (mediated by MG132) in cells expressing A53T a-syn and on a-syn oligomers (
ESCRT members have been implicated in neurodegenerative proteinopathies, such as Hrs of ESCRT-0 and Tsg101 of ESCRT-I in models of Alzheimer's disease (21). ESCRT controls formation of multivesicular bodies (MVBs), a subset of late endosomes that contain cargo-laden intralumenal vesicles (ILVs). MVBs can fuse with lysosomes leading to degradation of ILVs and their protein cargoes. In addition, ESCRT-III is important for lysosome maintenance and repair of lysosomal membranes (22). Mutations in the CHMP2B gene are rare but established causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (23, 24). A genome-wide association study has also nominated a CHMP2B variant as a genetic risk factor for PD (25). The CHMP2B protein is expressed in all neurons, and brains of mice expressing mutant CHMP2B form neuronal inclusions due to impaired lysosomal degradation (26). Furthermore, CHMP2B has been found to co-localize with a-syn in Lewy bodies in brains of PD patients (27).
CHMP2B has been reported to bind to Vacuolar Protein Sorting 4 (VPS4), an AAA-type adenosine triphosphatase (20). This interaction is mediated by the MIM on CHMP2B and a Microtubule Interacting and Trafficking (MIT) domain on VPS4. It was found that Pdpep1.3 inhibits the CHMP2B-VPS4 interaction (
Pdpep1.3 Promotes Degradation of a-Syn
Determination of the downstream effects of Pdpep1 were sought. Since ESCRT is involved in protein degradation, playing an important role in the endolysosomal pathway, it was hypothesized that Pdpep1.3 might promote degradation of a-syn. To test this, A53T a-syn protein levels in HEK293 cells co-transfected with Pdpep1 and its optimized versions was examined. Cells transfected with each of the peptides or with full-length CHMP2B demonstrated reduced a-syn protein levels by immunoblotting, while the control peptide, Scramble1.3 (same amino acid composition but scrambled sequence), had no effect (
Pdpep1.3 Disrupts a Newly Identified Interaction Between a-Syn and CHMP2B
Increased levels of a-syn have been reported to disrupt ESCRT and the endolysosomal pathway (30), but the exact mechanism of this disruption remains unknown. It was hypothesized that it could be mediated by a direct interaction between a-syn and CHMP2B; it has been suggested previously that a-syn may exert an effect on the endolysosomal pathway through CHMP2B (30). Co-immunoprecipitation experiments were first performed and it was found that a-syn interacts directly with CHMP2B (
Whether Pdpep1.3 could perturb the a-syn-CHMP2B interaction and thereby restore CHMP2B function in the ESCRT pathway was questioned. Pdpep1.3 directly interacted with CHMP2B in vitro with an affinity similar to the a-syn 103-114 peptide (
Pdpep1.3 Rescues Lysosomal Activity Disrupted by a-Syn
Since both ESCRT and a-syn can influence lysosomal function, assessment of the effect of Pdpep1.3 on lysosomes was sought. The levels of the lysosomal marker, Lysosomal-Associated Membrane Protein 1 (LAMP1) were first measured. Decreased LAMP1 is associated with accumulation of a-syn in brains of PD patients and rodent models (32). A similar decrease in LAMP1 with overexpression of A53T a-syn in HEK293 cells (
The effect of Pdpep1.3 on CD63, a marker of MVBs early in the endolysosomal pathway (33) was then assessed. Expression of A53T a-syn was associated with a substantial loss of CD63, consistent with disruption of MVB formation and the endolysosomal pathway, as indicated by the LAMP1 decrease. Co-expression with Pdpep1.3 resulted in the return of CD63, indicating the peptide rescued MVB formation (
Next, it was sought to directly test whether Pdpep1.3 affects lysosomal function. To this end, a lysosomal flux assay in which a self quenched substrate taken up by cells demonstrates a fluorescence signal proportional to intracellular lysosomal activity as the substrate is degraded (48) was used. As expected, it was found that A53T a-syn co-expressed with Scramble1.3 reduced lysosomal flux, consistent with a disruption of lysosomal activity (
Finally, it was sought to distinguish whether the degradation pathway disrupted by a-syn and rescued by Pdpep1.3 is autophagy-related (i.e., the autophagy-lysosomal pathway) or involves direct degradation by the lysosome without autophagosome involvement. To this end, p62, a prototypical autophagy receptor (34) was knocked down, and it was found that Pdpep1.3's effect on cell viability was not significantly affected, suggesting Pdpep1.3 acts independently of the autophagy-lysosomal pathway (
Pdpep1.3 Reduces a-Syn-Mediated Neurodegeneration In Vivo
Overexpression of WT or mutant a-syn in dopaminergic neurons causes degeneration of neurites and eventual loss of soma in several animal models (38). Such models were used to examine whether treatment with Pdpep1.3 could attenuate a-syn-mediated neurodegeneration in vivo.
First, Pdpep1.3 in C. elegans (
Second, the effect of Pdpep1.3 on a-syn oligomerization in an AAV vector-based rat model by directly co-injecting AAV-V1S and AAV-SV2 (a-syn fused to N- or C-terminal halves of YFP, respectively was determined; see above) in the substantia nigra (SN), as it was done previously (16), with AAVs that express Pdpep1.3 or Scramble1.3. At 6 weeks post-injection, a significant reduction in a-syn oligomer levels was observed, measured by YFP positive area, in dopaminergic neurons in the SN (
Third, a preclinical rat model of PD in which SN degeneration is induced by AAV vector-mediated expression of human mutant A53T a-syn (39-42) (
Pdpep1.3 Reduces a-Syn Accumulation in Human Cells with PD-Associated Mutations
Finally, to investigate the effects of Pdpep1.3 on endogenous a-syn in disease-relevant human models, cells with a-syn gene (SNCA) mutations known to cause PD were used. An autosomal dominant form of PD is caused by triplication mutation of SNCA with doubling in the effective load of the WT gene peripherally and in the brain (44). Skin fibroblasts from a PD patient with SNCA triplication and transduced them with AAVs expressing RFP-tagged peptides were cultured. It was found that Pdpep1.3 reduced a-syn fluorescence compared with Scramble1.3 control (
Further details of the methods and materials are provided in Example 3.
The results show that a-syn inhibits the endolysosomal pathway via interaction of its C-terminal region with CHMP2B. It is proposed that a-syn oligomers are instrumental in this inhibition since the C-terminal regions of multiple a-syn proteins are exposed in oligomers, thus avidity will lead to enhanced affinity of the a-syn-CHMP2B interaction. Moreover, this reduced lysosomal function will likely lead to reduced degradation of a-syn itself, thus resulting in further accumulation, which in turn leads to further lysosomal disruption. The findings thus illustrate a potential mechanism by which a-syn oligomerization can lead to severe failure of the cellular machinery.
The optimized peptide derived from the proteomic screens, Pdpep1.3, directly disrupts the a-syn-CHMP2B interaction, breaking the feedback loop and thereby restoring ESCRT function and lysosomal degradation. The net effect is an increase in lysosomal activity in neurons, increasing the clearance of a-syn, including a-syn oligomers. The effectiveness of Pdpep1.3 in promoting clearance of overexpressed a-syn both in vitro and in vivo has been demonstrated, and it has been shown that expression of Pdpep1.3 can reduce dopaminergic cell death in preclinical models of PD. also It has also been shown Pdpep1.3 to be efficacious at inhibiting a-syn accumulation and restoring the endolysosomal pathway in human PD models. This has potential important therapeutic implications as a peptide, such as Pdpep1.3, that facilitates a-syn degradation to reduce overall a-syn protein levels will circumvent many of the challenges faced by current approaches which depend on targeting specific a-syn conformations. Thus, targeting this novel pathogenic interaction between a-syn and CHMP2B may hold promise as a disease-modifying therapeutic strategy for the treatment of PD.
An optimized library based on the Pdpep1.3 peptide was designed in order to identify variants with higher activity. It is demonstrated that Pdpep1.3 peptide binds to CHMP2B and inhibits the CHMP2B-VPS4 interaction. Consequently, a I approach by building a combinatorial library of single mutants, and double mutants in positions I, i+4 was adopted. Also, extended and shortest variants of the Pdpep1.4 peptide were included by including amino acids of CHMP2B upstream and downstream of the natural sequence. Parent, WT asyn and A53T cell lines were used to screen for peptides that rescue cell viability, and the top 10 peptides are displayed in
One approach to overcome the typical limitations of peptide cellular uptake, half-life in blood, and BBB crossing efficiency is stapling the peptide by covalently linking the sidechains of two amino acids using an hexafluorobenzene, thereby forming a peptide macrocycle. Molecular dynamic simulations were performed to measure the stability of 5 different alternatives. The peptide structure was based on the C-terminal fragment of CHMP2B on the PDB 2JQK. The heaxaflurobenze was parametrized using amber tools (antechamber, and xleap) and gaussian. Each structure model was explicitly solvated by TIP3P water molecules in truncated octahedral periodic boundary conditions, and sodium counter ions were added for overall charge neutrality. Then, each system was minimized and equilibrated and a total of 100 ns of simulated were generated. Three out of the five were selected based on the performance during the simulations and the information about the potential hotspots in the sequence inferred and collected from all the experiments. The best candidates were DEEYCRQWKALC (SEQ ID NO: 35), DIEICFQLKALC (SEQ ID NO: 36) and DEEICRQLDALC (SEQ ID NO: 37) (Pdpep1.4 variants) (the C marks the residues modified for stapling-peptides to be chemically stapled are modified to introduce Cysteines at the locations to be stapled and these Cysteines are used for the chemical stapling). Optimized peptides DEEYCRQWKALC (SEQ ID NO: 35), DIEICFQLKALC (SEQ ID NO: 36) and DEEICRQLDALC (SEQ ID NO: 37) (Pdpep1.4 variants) showed increase in A53T cell viability (
Affinity measurements revealed that optimized macrocycles bind to truncated CHMP2B (1-199) as well as full length CHMP2B (
PCR primers that included the Illumina adaptor sequences were used to amplify peptide coding sequences recovered from selection and cell sorting. Results were demultiplexed, and peptide counts were tallied. After demultiplexing, only reads with an average quality Phred score of >30 were selected, and frequencies were calculated. Finally, the reads were then normalized to the total number of peptides read for that sample population, and a scalar factor was applied. In parallel, ˜25 individual colonies were Sanger sequenced to compare to the NGS data.
The identification of potential targets for the active peptides was conducted by mapping the sequences to the interacting interfaces of protein-protein interactions in the PDB. Multiple sequence mismatches were allowed, and a maximum of 3 mismatches were applied. The mapping complexes were ranked by sequence identity and the number of GO terms enriched in PD were annotated for the proteins involved in the PPI (Table 1). The enrichment term analysis was performed using DAVID based on the list of 330 genes annotated by the Parkinson's Disease Gene Ontology Annotation Institute at University College London. Next, how many of the Parkinson's standard GO terms were shared by the mapped proteins on each structure complex were counted. This record was used to rank and prioritize the matches. After visual inspection of a list of ten hits by discarding crystal packing contacts, and non-significant matches, the mapping of IPIQLKA (SEQ ID NO: 1) to the structure of the complex of a C-terminal fragment of CHMP2B binding to the MIT domain of VPS4B (PDB 2JQK) was selected. Both proteins belong to the ESCRT-III complex and play a vital role in vesicular body formation.
Overexpress® C41 E. coli cells were transformed by heat shock with 100 ng of pET-DEST42 CHMP2B vector and plated in LB plates containing 50 μg/ml of Carbenicillin for overnight incubation at 37° C. Transformed cells were grown in 500 ml of 2×YT media at 37° C. shaking at 220 rpm in a baffled 2 L flask. Cultures were grown until an OD of 0.6 was reached and then induced with 0.5 mM IPTG. After induction, cells were left shaking at 37° C. for a further 3 h at 220 rpm. Faster expression demonstrated reduced levels of non-specific truncations compared to overnight incubations at lower temperature. Cultures were pelleted at 3000×g and pellets were resuspended in 20 ml of BugBuster® Master Mix per 500 ml culture. The lysis reactions were incubated for 20 min at 4° C. mixing in a tube rotator. CHMP2B constructs expressed as inclusion bodies were insoluble in the BugBuster mix. The lysates were spun for 20 min at 3000×g to separate the inclusion bodies from the rest of the cell debris. Inclusion bodies were resuspended in 35 ml of 10 mM Phosphate pH 7.4, 150 mM NaCl, 6 M Guanidine HCl and spun at 34000×g at 4° C. for 20 min to remove lipidic contaminants. The supernatant was mixed with 5 ml of Ni-NTA resin in batch and incubated for 20 min in a tube rotator. Ni-NTA resins were pelleted by centrifugation at 270×g for 5 min and the supernatant was removed. The resins were washed three times with 10 mM Phosphate pH 7.4, 150 mM NaCl, 6 M Guanidine HCl, 30 mM Imidazole and proteins were eluted in 10 mM Phosphate pH 7.4, 150 mM NaCl, 6M Guanidine HCl, 500 mM Imidazole to a total volume of 10 ml. The eluted samples were dialysed overnight in 5 L of 50 mM Na Acetate pH 5.5 and 0.5 mM TCEP at 4° C. with a 10 kDa cutoff dialysis membrane. CHMP2B was further purified by Size exclusion chromatography with a HiLoad Superdex 16/60 S200 in an AKTA purifier. The column was pre-equilibrated in fresh 50 mM Na Acetate pH 5.5 and 0.5 mM TCEP, as CHMP2B displays greatly improved solubility in slightly acidic pH. CHMP2B eluted at the expected volume for a 26 kDa monomer. Samples were concentrated in a Amicon Ultra-15 spin concentrator to 0.5 mg/ml and stored at −80° C. Sample purity was confirmed by SDS-PAGE and protein identity was validated by ESI Mass Spectrometry.
VPS4B was expressed as a His-tagged Sumo construct in a pRSet B vector. Overexpress® C41 E. coli cells were transformed by heat shock with 100 ng of vector and cultures were grown as described for CHMP2B. Cultures were induced with 0.5 mM IPTG and incubated overnight at 20° C. Cells were pelleted as described and resuspended in PBS with a cOmplete Mini, EDTA-free protease inhibitor tablet. Cells were lysed by sonication in a Branson Digital Sonifier at 20% amplitude for 5 minutes in 10 s intervals between on and off sonication. The homogenized sample was spun at 34000×g at 4° C. for 20 min to remove insoluble contents. The soluble supernatant was incubated with 5 ml of Ni-NTA resin in batch for 20 min at 4° C. in a tube rotator. The resins were washed three times in PBS with 30 mM Imizadole and eluted in PBS with 300 mM imidazole. Imidazole was removed by dialyzing twice into 5 L of PBS.
To identify peptide inhibitors of protein-protein interaction, a previously designed human peptide library containing 50,549 heptamer C-terminal sequences, corresponding to 75,797 proteins, including isoforms and cleaved sequences was used. The oligonucleotide libraries were amplified and cloned into pLJM1 nGFP vector as previously described (12).
HEK293T cells, tet-off split luciferase a-syn cells, tet-off parent cells and SH-SY5Y cell lines were maintained in DMEM (ATCC) supplemented with 10% FBS and 1% pen/strep/glutamine, and the appropriate selection antibiotics when required. tet-off split luciferase a-syn cells and tet-off parent cells were kept with doxycycline at 1 ng/mL for inhibition of gene expression. SNCA triplication fibroblasts were obtained from NINDS (National Institute of Neurological Disorders and Stroke) and maintained in DMEM supplemented with 10% FBS and 1% pen/strep/glutamine. HA antibodies were obtained from Santa Cruz (7392) and Flag antibodies were purchased from Sigma (A8592). GFP antibodies were purchased from Abcam (ab290).
Lentiviruses were made in a 15 cm dish format by transfecting packaging cells (293T) with a three-plasmid system as previously described. Viral transduction of HEK293T cells, tet-off split luciferase a-syn cells, tet-off parent cells, and SH-SY5Y cells were performed with a multiplicity of infection of 0.3 (MOI=0.3). Infected cells were selected in puromycin-containing medium to eliminate uninfected cells and three aliquots of cells were collected for sampling of the initial (TO) cell population. Cells were treated with MG132 at concentrations of 0 μM, 10 μM, 25 μM or 50 μM. For the cell viability screen, surviving cells were collected and gDNA was extracted for identification of peptide inhibitors of cell toxicity. For the inhibition of a-syn aggregation screen, split YFP-a-syn constructs were transfected following stable expression of the peptides. Cells were sorted based on their GFP fluorescence intensities with a FACSVantage SE cell sorter (BD Bioscience).
Cells were harvested by trypsin treatment and centrifuged at 500×g for 5 minutes. The pellet was resuspended in ice-cold PBS and centrifuged again. The pellet was then resuspended at a concentration of 4×106 cells/ml in the sorting buffer, which is PBS containing 100 Kunitz Dnase l/ml, 10 μg/ml propidium iodide (Sigma) and 2% FBS. The sorting solution was also supplemented with either 10 μM forskolin, 100 μM 5,6-Dichlorobenzimidazole riboside (DRB, Sigma), 10 μM forskolin (Sigma) and 100 μM DRB or DMSO, as a control. The cells were then sent through a 40 μm filter to remove large clumps and loaded into either a FACScan Flow Cytometer (BD Bioscience) for cell analysis or a FACSVantage SE cell sorter (BD Bioscience) for cell sorting. The cells with positive propidium iodide staining (i.e., dead cells) were first eliminated from the analysis or sorting pool. For cell sorting, the desired population, either the most or least bright EGFP-positive cells, according to the purpose of the experiments (see Results), was sorted into either 15 ml conical tubes or 96-well plates, which both contained complete DMEM culture media.
Genomic DNA (gDNA) from peptide expressing cells at different time-points was extracted using QIAamp DNA Blood Mini Kit. PCR amplifications of peptides from gDNA in parallel with the lentiviral plasmid library (naïve library) were performed using indexed Illumina PCR primers to incorporate both the Illumina adapter sequences and indexing sequences. Each 50 μl reaction contained 3.2 μg of template, 2×PCR buffer, 2× enhancer solution, 300 μM each dNTP, 900 nM each of Adapter A (5′-AATGATACGGCGACCACCGAAATG-GACTATCATATGCTTACCGTAACTTGAA-3′) (SEQ ID NO: 42) and Adapter B (5′-CAAGCAGAA-GACGGCATACGATGTGGATGAATACTGCCATTTGTCTCGAGGTC-3′) (SEQ ID NO: 43), 1 mM MgSO4, 3.75 units of Platinum Pfx polymerase, and water to 50 μl. The PCR reaction was performed by denaturing at 94° C. for 5 minutes, followed by cycling (94° C. for 30 seconds, 65° C. for 30 seconds, 68° C. for 30 seconds)×28 cycles, 68° C. for 5 minutes, then cooling to 4° C. The resulting 244 bp product was purified by electrophoresis in 2% agarose followed by gel extraction. Peptide libraries were quantified using Quant-It assay (Invitrogen) and pooled. The insert size of the pooled library was confirmed on an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies), and the size corrected concentration was determined with RT-qPCR (KAPA biosystems Illumina standards). 11.4 pM of peptide library and 0.6 μM of PhiX control library (Illumina) were denatured and loaded on a HiSeq 2000 V3 150 cycle sequencing kit, with a read length of 150 bp.
Oligonucleotides encoding the specific peptides were synthesized and individually cloned into the pLJM1 nGFP lentiviral vector. Cells were infected with individual constructs, and cell viability was assessed using Cell Titer-Glo Luminescent assay (Promega) at 72 h post infection.
Cells were trypsinized from subconfluent cultures as described earlier, suspended in culture media, and then seeded into triplicate wells of a 96-well plate (100 μl well/1) at a density of 1.5×104 cells per well at standard culture conditions of 5% CO2 in air at 37° C. Cells were infected with lentivirus expressing peptide at an MOI of 5 for 72 h or transfected with plasmid for 72 h. Cell Titer-Glo reagent was added to each well (30 μL), according to the manufacturer's protocol and optical density of the plate was measured at 540 and 630 nm with a standard spectrophotometer.
Infected or transfected cells were scraped from 6-well dishes and lysed with lysis buffer (50 mM Tris-HCl pH7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1× protease inhibitor mixture (Sigma)) for 30 min at 4° C. The insoluble pellet was removed following a 10,000 rpm spin for 5 minutes at 4° C. Lysates were analyzed by SDS-PAGE/immunoblot using 4-20% Mini-PROTEAN Tris-glycine gels (Bio-Rad) transferred to PVDF membranes and blocked in 5% milk containing PBS-Tween-20 (0.1%) for 1 h. PVDF membranes were then incubated with specified primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and detected using enhanced chemiluminescence (GE Healthcare).
Infected neurons were scraped from 6-well dishes and lysed with RIPA buffer containing protease inhibitor cocktail (Roche). The Triton X-100 soluble fraction was then separated from the insoluble pellet by centrifugation. Protein concentration was quantified using the DC protein assay (BioRad). For each condition, 20 μg of protein lysate was run on 4-15% acrylamide gels (BioRad) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 5% skim milk in TBS+0.01% Tween-20 (TBS-T) for 1 h prior to incubation with primary antibody overnight at 4° C. Blots were subsequently washed 3 times in TBS-T for 10 minutes per wash, incubated in species specific secondary antibody for 1 h at 21° C., washed again, and then developed using ECL immunoblotting substrate (Pierce) and visualized on HyBlot CL autoradiographic film (Denville Scientific).
HEK293T cells were co-transfected with Flag-tagged target protein, HA-tagged source protein, and GFP-tagged peptide or GFP. Cells were lysed 48 h after transfections with radioimmune precipitation assay buffer (50 mM Tris-HCl pH7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 10 mM sodium pyrophosphate, 25 mM NaF, 1× protease inhibitor mixture (Sigma)) for 30 min at 4° C. and coimmunoprecipitated with Flag beads (Clontech). The resulting immunocomplexes were analyzed by immunoblot using the appropriate antibodies. Protein samples were separated using 4-20% Mini-PROTEAN Tris-glycine gels (Bio-Rad) transferred to PVDF membranes and blocked in 5% milk containing PBS-Tween-20 (0.1%) for 1 h. PVDF membranes were then incubated with specified primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and detected using enhanced chemiluminescence (GE Healthcare).
Purification of a-syn
Glycerol stocks were plated on LB plates containing 50 μg/ml of kanamycin for overnight incubation at 37° C. Bacterial cultures were grown in 500 ml of LB media at 37° C. with shaking at 220 rpm in a baffled 2 L flask. Cultures were grown until an OD of 0.6 was reached and then induced with 0.5 mM IPTG. After induction, cells were incubated at 37° C. for a further 2.5 h shaking at 220 rpm. Cells were harvested by splitting the 500 ml culture into two 250 ml conical tubes and spinning down at 4000× g for 15 min at 4° C. Cells were re-suspended in lysis buffer (50 mM KH2PO4, 400 mM NaCl, 100 mM KCl, 30 mM Imidazole, 10% v/v glycerol, 0.5% v/v Triton X-100) and lysed by 3 cycles of flash freezing on an ethanol/dry-ice mix for 12 mins, followed by heating in a water bath for 12 mins. The resultant mixture was spun down at 4000× g for 15 min at 4° C. and the supernatant was added directly to a His-spin trap (GE Healthcare) as per the manufacturer's instructions. The eluted product was dialyzed against PBS overnight, aliquoted at a concentration of 1 mg/ml and stored at −80° C. until ready for use.
Fluorescence polarization assays were carried out in 384-well black non-binding plates (Greiner 781906) in a Pherastar plate reader (BMG) with a Fluorescence Polarization Module 485-520-520. All peptides were synthesized by LifeTein with N-terminal FITC moieties. Binding assays were performed in 50 mM Na Phosphate pH 5.5, 0.5 mM TCEP, 0.005% Triton-X100 (2) or in 20 mM Na Acetate pH 5.5, 0.5 mM TCEP, 0.005% Triton-X100 as indicated. FITC-labelled peptides were kept at a constant 50 nM concentration and CHMP2B was serially diluted 1:1 starting from 30 μM (0.5 mg/ml).
The a-syn competition assay had a constant 50 nM concentration of FITC-Pdpep1.3 peptide and 1 μM of CHMP2B; a-syn was serially diluted 1:1 starting from 30 μM. Plates were incubated for 30 min at room temperature before reading. Raw FP data in millipolarization (mp) units was fitted to the following equation in Graphpad PRISM 8:
tet-off cells stably expressing split luciferase a-syn constructs were trypsinized from subconfluent culture and seeded in a 96-well plate at a density of 15,000 cells per well. Cells were incubated overnight at 37° C. in 5% CO2. Cells were transfected with GFP-peptide plasmids. After 6 h of incubation, 20 μL of cell medium was transferred to a black flat-bottomed 96-well plate. 50 μL of Working solution (Pierce Gaussia-Firefly Luciferase Dual Assay Kit, Thermo Scientific #16181) was added into each well containing cell medium. Immediately after adding the reagent, samples were read using a luminometer with a 480 nm filter.
The Lysosome Intracellular Activity Assay kit was purchased from Abcam and used per manufacturer's instructions (ab234622). Briefly, cell medium was replaced with fresh medium containing DMSO, Leupeptin, or Bafilomycin A1 and incubated for 1 h at 37° C. with 5% CO2. Self-quenched substrate was added into each condition and incubated for 1 h (HEK293 cells) or overnight (SNCA triplication fibroblasts) at 37° C. with 5% CO2. After incubation, cells were washed twice in ice-cold 1× assay buffer containing DMSO, Leupeptin, or Bafilomycin A1. For HEK293 cells, cells were re-suspended in PBS containing DMSO or Leupeptin and analyzed by FACS. For fibroblasts, cells were incubated in PBS containing DMSO or Bafilomycin A1 and imaged by confocal microscopy.
Adeno-associated virus (AAV) of a % serotype was used to express A53T a-syn (AAV-A53T), a truncated version of A53T a-syn lacking amino acids 103-140 (Aa-syn(103-140)), and WT a-syn, fused to either the N-terminus half of YFP (AAV-V1S) or the C-terminus half of YFP (AAV-SV2), all under the control of the CAG promoter, a hybrid of the chicken beta actin (CBA) promoter fused with the cytomegalovirus (CMV) immediate early enhancer sequence (GeneDetect Ltd.), as previously described (3). An AAV1/2 vector lacking the A53T a-syn open reading frame was used as an empty vector control (AAV-Empty) for A53T a-syn experiments and an AAV1/2 vector expressing full length YFP was used as a control for experiments measuring a-syn oligomer formation.
Pregnant rats (E17) of the Sprague-Dawley strain were purchased from Envigo. Embryos were surgically removed from the mothers and cortices dissected in Hanks Balanced salt solution (Gibco). The meninges were removed, and cells were dissociated using a papain dissociation system (Worthington) before being resuspended in Neurobasal medium A supplemented with antibiotic-antimycotic solution (Gibco), L-glutamine substitute (GlutaMAX™; Gibco) and factor B27 (Gibco). Cells were plated on poly-D-lysine coated glass coverslips at a density of 5×105 cells/well, or on poly-D-lysine coated 6-well cell culture plates at a density of 2×106 cells/well, and incubated at 37° C. in 5% CO2 with half media changes every 3 days. Cells were transduced with AAVs 2 days post-isolation at a multiplicity of infection (MOI) of 3000. Media containing AAV vectors were removed after 72 h and cells were fixed with 4% PFA for immunofluorescence staining or lysed for immunoblotting at 8 days post-isolation.
Human iPSC-Derived Dopaminergic Neuron Culture
iCell Dopa neurons (A53T a-syn mutant and isogenic control) were purchased from Fujifilm Cellular Dynamics (Madison, WI, USA) and cultured as per manufacturer's instructions. Briefly, cells were plated in complete maintenance medium provided by the manufacturer on poly-L-ornithine/laminin coated plates (ibidi). Cells recovered from thawing for 24 h and then were transduced with AAV-Scramble1.3-RFP or AAV-Pdpep1.3-RFP for 48 h at which time media was completely changed. Cells were fixed using 4% PFA at 7 days post-plating.
After fixation, cells were permeabilized with 0.2% Triton X-100 for 15 min, washed 3 times with PBS and then incubated with blocking solution (1% BSA, 22.52 mg/mL glycine, 0.1% Tween-20 in PBS) for 1 h. Primary antibodies were diluted in blocking solution and incubated overnight at 4° C. Following three washes with PBS, cells were next incubated with fluorescent secondary antibodies diluted in blocking solution for 1 h at room temperature. Following another 3 PBS washes, nuclei were counterstained with DAPI (ThermoFisher) and then coverslips were mounted on slides using fluorescence mounting medium (DAKO) and sealed using clear nail varnish.
C. elegans Strains
C. elegans strains were grown and maintained under standard conditions at 22-23° (4). BZ555 [dat-1p::gfp] was obtained from the C. elegans Genetics Center (CGC; University of Minnesota, St Paul, MN, USA), TWH1 ([dat-1p::a-syn(A30P), ges-1p::DsRed]; [dat-1p::gfp]) was obtained by crossing BZ555 with A30P a-syn transgenic animals (kindly provided by Dr. Takeshi Iwatsubo, University of Tokyo) (5).
C. elegans Neurite Length Assay
The peptide construct (pPD97.78_osm-6p_TagRFP_wpeptide) was obtained by subcloning the following fragments into NheI and SpeI sites of pPD97.78 (A. Fire): NheI-AgeI osm-1p fragment (2.4 kb) obtained by PCR using primer set 5′-catccgctagcggatcccatggccagtggaatcacc-3′ (SEQ ID NO: 44) and 5′-ccataccggtagatgtatactaatgaaggtaatagcttgaaagag-3′ (SEQ ID NO: 45) and N2 genomic DNA as a template, AgeI-EcoRI TagRFP fragment obtained by PCR using primer set 5′-gttgaccggtATGGTGTCTAAGGGCGAAGAGCTG-3′ (SEQ ID NO: 46) and 5′-ggcagaattcgaATTAAGTTTGTGCCCCAGTTTGCTAGG-3′ (SEQ ID NO: 47), EcoRI-SmaI peptide (FEELEAQLARLR (SEQ ID NO: 48)) fragment digested from CMV_FDDLEAQLARLR_worm, SmaI-SpeI unc-54 3′-UTR fragment digested from pPD95.75 (Fire vector). The TagRFP control construct was obtained by replacing the EcoRI-SmaI peptide fragment and SmaI-SpeI unc-54 3′-UTR fragment from pPD97.78_osm-6p_TagRFP_wpeptide, to EcoRI-SpeI unc-54 3′-UTR fragment digested from pPD95.75 (6).
Each plasmid (40 ng/μl) was injected with a co-injection marker, sur-5p::mCherry into TWH1. The animals carrying the extrachromosomal arrays were transferred to new plates, and their progenies were analyzed at the adult stage under a widefield microscope (Zeiss AxioObserver). Pearson Chi-Square test for pairwise comparison of neurite length frequencies was used for statistical analysis.
Adult female Sprague-Dawley rats (250-280 g; Envigo) were pair-housed in cages with wood bedding and had access to food and water ad libitum. The animal colony was maintained in a regular 12-h light/dark cycle. All procedures were approved by the University Health Network Animal Care Committee in accordance with guidelines and regulations set by the Canadian Council on Animal Care.
Animals were secured in a stereotactic frame under isoflurane/oxygen anaesthesia and ketoprofen (5 mg/kg) analgesia. The surgical site was shaved and sterilized with iodine/betadine prior to making a 2 cm incision along the midline. The scalp was exposed and a unilateral injection targeting the SN was performed at coordinates AP−5.2 mm, ML−2 mm and DV−7.4 mm with respect to the bregma as a point of reference. For each animal, a total volume of 2 μl of virus was injected at a rate of 0.5 μl/min using a microinjection pump and 10 μl Hamilton syringe with a 26-gauge needle. For A53T groups, 1 μl (low dose) or 1.34 μl (high dose) of AAV1/2-A53T a-syn (5.1×1012 genomic particles/ml), 0.14 μl of AAV1/2-Pdpep1.3-GFP or scramble1.3-GFP (5.1×1012 genomic particles/ml) and 0.86 μl or 0.52 μl of sterile PBS was injected; for EV groups, 1 μl (low dose) or 1.34 μl (high dose) of AAV1/2-EV (5.1×1012 genomic particles/ml) replaced AAV1/2-A53T. For V1S+SV2 groups, 0.58 μl of AAV1/2-V1S (1.1×1012 genomic particles/ml), 0.58 μl of AAV1/2-SV2 (1.1×1012 genomic particles/ml), 0.14 μl of AAV1/2-Pdpep1.3-RFP or scramble1.3-RFP (5.1×1012 genomic particles/ml) and 0.7 μl of sterile PBS was injected; for the YFP groups, 1.16 μl of AAV1/2-YFP (1.1×1012 genomic particles/ml), 0.14 μl of AAV1/2-Pdpep1.3-RFP or scramble1.3-RFP (5.1×1012 genomic particles/ml) and 0.7 μl of sterile PBS was injected. At the end of virus injection, the needle remained in place for 5 minutes before gradual removal.
Spontaneous forepaw use was evaluated using the cylinder test 1 day prior to stereotactic AAV injection, at 21 days post-injection and at 41 days post-injection. Following overnight food restriction, individual rats with right paws marked black were placed into a glass cylinder in front of two mirrors and videos recorded. An observer blinded to treatment conditions later scored the videos by recording whether animals used their left or right forepaw to touch the inner glass surface upon rearing. A total of 5 min of video recording was scored and a minimum of 10 total touches was required for data inclusion (61).
Animals were uthanized by transcardial perfusion with heparinised saline under isoflurane/oxygen anaesthesia. Brains were then removed and the ventral part, including the ventral striatum, was snap frozen in liquid dry ice-cooled isopentane. A single 1 mm thick section of the ventral striatum was immediately cut, using a matrix, for high-performance liquid chromatography (HPLC) analysis of biogenic amines. The dorsal part, including the dorsal striatum, STN, and the SN, was immersion-fixed in 4% paraformaldehyde in 0.1M PBS for 2 days and cryo-protected in 30% sucrose in 0.1M PBS solution for another 3 days until the brains sank. For immunofluorescent staining, 40 μm coronal cryosections were then prepared using a sliding microtome (Leica Microsystems Inc.) and 6 series of sections were stored in cryoprotectant (30% glycerol, 30% ethylene glycol, 40% PBS) at −20° C. until use.
Immunofluorescence staining for a-syn, tyrosine hydroxylase and LAMP1 was performed by washing free-floating sections with PBS-T (0.2% Triton X-100) three times for 10 min each at room temperature. Sections were then incubated in blocking solution (1% BSA, 10% normal goat serum in PBS-T) for 1 h. After blocking, sections were incubated with primary antibodies in antibody solution (2% normal goat serum in PBS-T) overnight at RT. Sections were then washed in PBS and incubated with secondary antibodies diluted in antibody solution for 1 h in the dark at room temperature. Sections were then mounted onto glass slides and allowed to dry and then fluorescence mounting medium (DAKO) was applied followed by cover slips.
Confocal images of immunofluorescent staining were acquired with a Zeiss LSM880 confocal microscope equipped with 405, 488, 555, and 639 nm laser lines. All images were taken within the linear range at constant gain and pinhole settings at optimal resolution settings determined by the software. For primary cortical neurons, the software was programmed to acquire an image every 1 μm for a total of 11 μm, capturing all of the neurons visible in the z-plane in each field of view using a 63× objective. For animal experiments, the whole midbrain or striatum regions were imaged using a 10× objective. Ten serial coronal midbrain sections were imaged per animal, separated by 240 μm intervals. 3-6 images of the striatum per animal were acquired and a representative image of a single coronal section present in all sets was chosen for analysis, based on anatomical features.
Confocal images of immunofluorescent staining of midbrain and striatal sections were processed using HALO software (Indica Labs), which is a well validated tool for automatic quantification of neurons in brain tissue sections (8-10). Initially, ipsilateral SN was selected as a region of interest (ROI). Dopaminergic neurons were subsequently identified by automated detection of TH-labelled objects within this ROI, as previously validated by correlation analyses with traditional stereological methods (11, 12). Levels of total a-syn were assessed in this ROI by measuring the area and intensity of anti-a-syn staining.
Confocal images of primary cortical neurons, dopaminergic neurons, and LAMP1 staining within the SN were processed using Imaris software (Oxford instruments). Z stacks were projected to give a 3D reconstruction of the field of view and mean pixel intensity per GFP+ cell (or RFP+ cell for V1S/SV2 experiments) was calculated for a-syn or LAMP1 signal using the software's surfaces module. For LAMP1 staining within the SN, the number of LAMP1+ puncta within GFP+ cells was calculated for each field of view; 3 fields of view were imaged/animal.
HPLC was performed as described (13). The investigator was blinded to experimental groups and treatment conditions. Brain sections were homogenized followed by centrifugation at 10,000×g for 20 minutes. Catecholamines were determined from the supernatant. Values of catecholamines are expressed as ng analyte/mg total protein.
All data are represented as mean±s.d. with at least 3 independent experiments, unless otherwise stated. Statistical analysis was performed using GraphPad Prism 8.
Peptides were purchased from LifeTein. Cyclic angio-peptides were synthesized as follow (N-Terminal: FITC-Ahx, C-Terminal: Amidation, Disulfide Bridge between cysteine residues). Non-cyclic angio-peptides were synthesized as follow (N-Terminal: FITC-Ahx, C-Terminal: Amidation). For the cyclic bisphenyl peptide, (N-Terminal: FITC-Ahx, C-Terminal: Amidation, Perfluoroarene-based stapling on both cysteine residues using Decafluorobiphenyl).
Description: Cell viability effect of peptides in A53T expressing HEK293 cells at different concentrations of cyclized and non-cyclized angio-conjugated peptides and cyclic bisphenyl conjugated peptides was tested. The results are shown in
While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.
The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.
This application claims the benefit of priority to U.S. Provisional Application No. 63/289,912, filed Dec. 15, 2021, the contents of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051837 | 12/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63289912 | Dec 2021 | US |
