Patent Application
20220072097
Applicant submits herewith a Sequence Listing in computer readable form and in compliance with 37 C.F.R. §§ 1.821-1.825. This sequence listing is in ASCII TXT format with filename “10336-185US2_2021_09_23_Sequence_Listing,” a 200,001 bytes file size, and creation date of May 12, 2017. The content of the Sequence Listing is hereby incorporated by reference.
Alzheimer's disease (AD) is the most common form of dementia, and its risk accelerates after age 65. With a rapidly expanding aging population, AD is projected to become an overwhelming medical burden to the world.
A definitive pathological hallmark of Alzheimer's disease (AD) is the progressive aggregation of β-amyloid (Aβ) peptides in the brain, a process also known as β-amyloidosis, which is often accompanied by neuroinflammation and formation of neurofibrillary tangles containing Tau, a microtubule binding protein_1.
Evidence from human genetic studies showed that overproduction of Aβ due to gene mutations inevitably inflicts cascades of cytotoxic events, ultimately leading to neurodegeneration and decay of brain functions. Cerebral accumulation of Aβ peptides, especially in their soluble forms, is therefore recognized as a key culprit in the development of AD1. In the brain, Aβ peptides mainly derive from sequential cleavage of neuronal Amyloid Precursor Protein (APP) by the β- and γ-secretases. However, despite decades of research, molecular regulation of the amyloidogenic secretase activities remains poorly understood, hindering the design of therapeutics to specifically target the APP amyloidogenic pathway.
Pharmacological inhibition of the β- and γ-secretase activities, although effective in suppressing Aβ production, interferes with physiological function of the secretases on their other substrates. Such intervention strategies therefore are often innately associated with untoward side effects, which have led to several failed clinical trials in the past2-4. To date, no therapeutic regimen is available to prevent the onset of AD or curtail its progression.
Besides Aβ, Tau is another biomarker that has been intensively studied in AD. Cognitive decline in patients sometimes correlates better with Tau pathology than with Aβ burden5,6. Overwhelming evidence also substantiated that malfunction of Tau contributes to synaptic loss and neuronal deterioration7.
In addition to AD, many other neurodegenerative diseases also involves Aβ or Tau pathologies, and there is no disease modifying therapy available for any of these debilitating diseases.
Disclosed herein are peptides, compositions, and methods to treat and prevent neurodegenerative diseases that involve β-amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt-Jakob disease.
These peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in at-risk subjects, such as people with Down syndrome and those who have suffered from brain injuries or cerebral ischemia, as well as the aging population.
In some embodiments, the disclosed peptides, compositions, and methods disrupt the binding between Protein Tyrosine Phosphatase sigma (PTPσ) and APP, preventing β-amyloidogenic processing of APP as well as Tau aggregation.
In some embodiments, the disclosed compositions and methods restore the physiological balance of two classes of PTPσ ligands in the brain microenvironment, namely the chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS), and thereby prevent abnormally increased β-amyloidogenic processing of APP.
Unlike the anti-Aβ antibodies in current clinical trials that passively clear β-amyloid, the therapeutic strategy disclosed herein inhibits the process upstream of β-amyloid production. Unlike the β- and γ-secretase inhibitors in current clinical trials, the therapeutic strategy disclosed herein inhibits β-amyloid production without affecting other major substrates of these secretases. Therefore the strategy disclosed herein may be more effective with fewer side effects compared to the most advanced AD drug candidates in clinical trials.
Disclosed herein is a peptide for treating or preventing the aforementioned neurodegenerative disorders, the peptide comprising a decoy fragment of APP, a decoy fragment of PTPσ, or a combination thereof. In some embodiments, the decoy fragment of APP is a peptide comprising at least 5 consecutive amino acids of SEQ ID NO:1. In some embodiments, the decoy fragment of APP is a peptide comprising at least 10 consecutive amino acids of SEQ ID NO:1. For example, the decoy fragment of APP can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:112, SEQ ID NO:139, SEQ ID NO:151, SEQ ID NO:157, SEQ ID NO:251, SEQ ID NO:897. In some embodiments, the decoy fragment of PTPσ is a peptide comprising at least 4 consecutive amino acids of SEQ ID NO:442. For example, the decoy fragment of PTPσ can comprises the amino acid sequence SEQ ID NO:655, SEQ ID NO:769, SEQ ID NO:898, or SEQ ID NO:899. In some embodiments, the peptide further comprises a blood brain barrier penetrating sequence. For example, the blood brain barrier penetrating sequence comprises amino acid sequence SEQ ID NO: 880, SEQ ID NO: 883, SEQ ID NO: 888, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896.
Also disclosed is a method that restores the physiological molecular CS/HS balance that may be used to treat and prevent aforementioned neurodegenerative diseases. In some embodiments, administering HS, or its analog heparin, or their mimetics modified to reduce anti-coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the CS/HS balance. In some embodiments, the physiological molecular CS/HS balance is restored by administering enzymes that digest CS (such as Chondroitinase ABC, also known as ChABC) or prevent HS degradation (such as Heparanase inhibitors PI-88, OGT 2115, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of PTPσ clustering8, such as multivalent antibodies, could be administered.
Also disclosed is a method of treating a neurodegenerative disorder in a subject, the method comprising administering to the subject an aforementioned composition or combination of compositions. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt-Jakob disease. In some embodiments, subjects are selected from at-risk populations, such as the aging population, people with Down syndrome, and those suffered from brain injuries or cerebral ischemia, to prevent subsequent onset of neurodegenerative diseases.
Also disclosed is a method of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration. In some embodiments, the method comprises providing a sample comprising APP and PTPσ in an environment permissive for APP-PTPσ binding, contacting the sample with a candidate compound, and assaying the sample for APP-PTPσ binding, wherein a decrease in APP-PTPσ binding compared to control values is an indication that the candidate agent is effective to slow, stop, reverse, or prevent neurodegeneration. In some embodiments, the method comprises contacting/incubating a candidate compound with cell membrane preparations extracted from fresh rodent brain homogenates, wherein a decrease in APP β- and/or γ-cleavage products is an indication that the candidate agent has the potential to slow, stop, reverse, or prevent neurodegeneration.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Experimental results in Example 1 show that neuronal receptor PTPσ mediates both β-amyloid and Tau pathogenesis in two mouse models. In the brain, PTPσ binds to APP. Depletion of PTPσ reduces the affinity between APP and β-secretase, diminishing APP proteolytic products by β- and γ-cleavage without affecting other major substrates of the secretases, suggesting a specificity of β-amyloidogenic regulation. In human APP transgenic mice during aging, the progression of β-amyloidosis, Tau aggregation, neuroinflammation, synaptic loss, as well as behavioral deficits, all show unambiguous dependency on the expression of PTPσ. Additionally, the aggregates of endogenous Tau are found in a distribution pattern similar to that of early stage neurofibrillary tangles in Alzheimer brains. Together, these findings unveil a gatekeeping role of PTPσ upstream of the degenerative pathogenesis, indicating a potential for this neuronal receptor as a drug target for Alzheimer's disease.
Experimental results in Example 2 show that two classes of PTPσ ligands in the brain microenvironment, CS and HS, regulate APP amyloidogenic processing in opposite manners. CS increases APP n-cleavage products, whereas HS decreases APP n-cleavage products. Because CS and HS compete to interact with receptor PTPσ yet lead to opposite signaling and neuronal responses, the ratio of perineuronal CS and HS is therefore crucial for the downstream effects of PTPσ and maintaining the health of the brain.
Experimental results in Example 3 further define that the binding between APP and PTPσ is mediated by a fragment on APP between its E1 and E2 domain and the IG1 domain of PTPσ.
The findings that PTPσ plays a pivotal role in the development of β-amyloid and Tau pathologies indicate that peptides, compositions, and methods disclosed herein may be suitable to treat and prevent neurodegenerative diseases that involve β-amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt-Jakob disease.
Additionally, these peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in at-risk populations, such as subjects with Down syndrome and those suffered from brain injuries or cerebral ischemia, as well as the aging population.
As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The term “protein” includes amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and can contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. The term also includes peptidomimetics and cyclic peptides.
As used herein, “peptidomimetic” means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetics typically enhance some property of the original peptide, such as increase stability, increased efficacy, enhanced delivery, increased half life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Pat. Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include β-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine, N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.
A “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.
As used herein, protein “binding” is the binding of one protein to another. The binding may comprise covalent bonds, protein cross-linking, and/or non-covalent interactions such as hydrophobic interactions, ionic interactions, or hydrogen bonds.
The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.
“Amyloid precursor protein” (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is cleaved by beta secretase and gamma secretase to yield Aβ. Amyloid beta (Aβ) denotes peptides of 36-43 amino acids that are involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. Aβ molecules cleaved from APP can aggregate to form flexible soluble oligomers which may exist in various forms. Certain misfolded oligomers (known as “seeds”) can induce other Aβ molecules to also take the misfolded oligomeric form, leading to a chain reaction and buildup of amyloid plaques. The seeds or the resulting amyloid plaques are toxic to cells in the brain.
“Protein tyrosine phosphatases” or “receptor protein tyrosine phosphatases” (PTPs) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins. Protein tyrosine phosphorylation is a common post-translational modification that can create novel recognition motifs for protein interactions and cellular localization, affect protein stability, and regulate enzyme activity. As a consequence, maintaining an appropriate level of protein tyrosine phosphorylation is essential for many cellular functions. Tyrosine-specific protein phosphatases catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are key regulatory components in many signal transduction pathways (such as the MAP kinase pathway) that underlie cellular functions such as cell cycle control/proliferation, cell death, differentiation, transformation, cell polarity and motility, synaptic plasticity, etc.
The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. An “at-risk” subject is an individual with a higher likelihood of developing a certain disease or condition. An “at-risk” subject may have, for example, received a medical diagnosis associated with the certain disease or condition.
“Tau proteins” (or τ proteins) are proteins that stabilize microtubules. They are abundant in neurons of the central nervous system and are less common elsewhere, but are also expressed at very low levels in CNS astrocytes and oligodendrocytes. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and other tauopathies are associated with tau proteins that have become defective, misfolded, tangled, and no longer stabilize microtubules properly.
The term “protein fragment” refers to a functional portion of a full-length protein. For example, a fragment of APP or PTPσ may be synthesized chemically or biologically for the purposes of disrupting the binding between APP and PTPσ. Such fragments could be used as “decoy” peptides to prevent or diminish the actual APP-PTPσ binding interaction that results in β-cleavage of APP and subsequent AO formation.
The phrase “functional fragment” or “analog” or mimetic of a protein or other molecule is a compound having qualitative biological activity in common with a full-length protein or other molecule of its entire structure. A functional fragment of a full-length protein may be isolated and attached to a separate peptide sequence. For example, a functional fragment of a blood-brain barrier penetrating protein may be isolated and attached to the decoy peptide that disrupts APP-PTPσ binding, thereby enabling the hybrid peptide to enter the brain and disrupt APP-PTPσ binding. Another example of a functional fragment is a membrane penetrating fragment, or one that relays an ability to pass the lipophilic barrier of a cell's plasma membrane. An analog of heparin, for example, may be a compound that binds to a heparin binding site.
As used herein, “cyclic peptide” or “cyclopeptide” in general refers to a peptide comprising at least one internal bond attaching nonadjacent amino acids of the peptide, such as when the end amino acids of a linear sequence are attached to form a circular peptide.
The term “antibody” refers to natural or synthetic antibodies that selectively bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.
As used herein, “enzyme” refers to a protein specialized to catalyze or promote a specific metabolic reaction.
“Neurodegenerative disorders” or “neurodegenerative diseases” are conditions marked by the progressive loss of structure or function of neural cells, including death of neurons and glia.
The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
The term “administering” refers to an administration that is intranasal, oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
The term “pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical use. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further below. The pharmaceutical compositions also can include preservatives. A “pharmaceutically acceptable carrier” as used in the specification and claims includes both one and more than one such carrier.
The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions (“conservative variant”), non-conservative amino acid subsitutions (e.g., a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, or 95% homology to a reference sequence.
The term “percent (%) sequence identity” or “homology” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
Compositions
Disclosed herein are peptides for treating and preventing the aforementioned neurodegenerative diseases, such as Alzheimer's disease. In some embodiments, the peptides disrupt the binding between PTPσ and APP, preventing β-amyloidogenic processing of APP without affecting other major substrates of the β- and γ-secretases. The peptide may be a decoy fragment of APP, a decoy fragment of PTPσ, or a combination thereof.
In some embodiments, a decoy peptide could be fabricated from the PTPσ-binding region on APP, which is the fragment between its E1 and E2 domains (SEQ ID NO:1). In some embodiments, a decoy peptide could be fabricated from the APP-binding region on PTPσ, which is its IG1 domain (SEQ ID NO: 442). In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP E2 domain or a fragment thereof. In some embodiments, a decoy peptide could be fabricated that corresponds to the entire APP E1 domain or a fragment thereof. In some embodiments, a PTPσ peptide is used in combination with an APP peptide.
In some embodiments, the peptide is a fragment of the PTPσ-binding domain of APP. Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO:1, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof.
Therefore, in some embodiments, the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 24 consecutive residues of SEQ ID NO: 1, or from the group consisting of the below:
In some embodiments, the peptide is a fragment of the APP-binding domain of PTPσ. Therefore, in some embodiments, the peptide is a fragment of SEQ ID NO:442, as listed below, which has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids, or a conservative variant thereof. The underlined amino acids represent residues in the ligand-binding pocket.
Therefore, in some embodiments, the peptide comprises an amino acid sequence selected from 10 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 11 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 12 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 13 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
In some embodiments, the peptide comprises an amino acid sequence selected from 14 consecutive residues of SEQ ID NO: 442, or from the group consisting of the below:
In some embodiments, the disclosed peptide further comprises a blood brain barrier penetrating sequence. For example, cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). The cellular internalization sequence can be any cell-penetrating peptide sequence capable of penetrating the BBB. Non-limiting examples of CPPs include Polyarginine (e.g., R9), Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 1).
Therefore, in some embodiments, the disclosed peptide is a fusion protein, e.g., containing the APP-binding domain of PTPσ, the PTPσ-binding domain of APP, or a combination thereof, and a CPP. Fusion proteins, also known as chimeric proteins, are proteins created through the joining of two or more genes, which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with function properties derived from each of the original proteins. Recombinant fusion proteins can be created artificially by recombinant DNA technology for use in biological research or therapeutics.
In some embodiments, linker (or “spacer”) peptides are also added which make it more likely that the proteins fold independently and behave as expected. Linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents which enable the liberation of the two separate proteins. This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide, or a hexa-his peptide (aka: a 6×his-tag) which can be isolated using nickel or cobalt resins (affinity chromatography). Chimeric proteins can also be manufactured with toxins or antibodies attached to them in order to study disease development.
Compositions that Restore Molecular Balance of CS and HS in the Perineuronal Space:
Chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS) are two main classes of glycosaminoglycans (GAGs) in the brain that are sensed by neurons via Receptor Protein Tyrosine8. The ratio of CS and HS therefore affects the downstream effects of PTPσ, because CS and HS compete to interact with the receptor yet lead to opposite signaling and neuronal responses (such as neurite regeneration). CS increases but HS decreases APP β-cleavage products (Example 2). Therefore, methods involving administering to the subject a composition that restore the physiological molecular CS/HS balance may be used to treat and prevent aforementioned neurodegenerative diseases. These therapies could be applied alternatively or in addition to the polypeptides listed above. In some embodiments, administering HS, or its analog heparin, or their mimetics modified to reduce anti-coagulant effect, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the physiological molecular CS/HS balance. In some embodiments, the balance is restored by administering enzymes that digest CS (such as ChABC) or prevent the degradation of HS (such as Heparanase inhibitors PI-88, OGT 2115, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of PTPσ clustering8, such as multivalent antibodies, could be administered.
Pharmaceutical Compositions
The peptides disclosed can be used therapeutically in combination with a pharmaceutically acceptable carrier. Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
In some embodiments, the peptides described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (See, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, 4th Edition, 1985, 126).
Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%-100% active ingredient, or in one embodiment 0.1-95%.
Methods of Screening
Also disclosed are methods of screening for candidate compounds that slow, stop, reverse, or prevent neurodegeneration.
In some embodiments, the method comprising providing a sample comprising APP and PTPσ in an environment permissive for APP-PTPσ binding, contacting the sample with a candidate compound, and assaying the sample for APP-PTPσ binding, wherein a decrease in APP-PTPσ binding compared to control values is an indication that the candidate agent is effective to slow, stop, reverse, or prevent neurodegeneration.
The binding of PTPσ to APP can be detected using routine methods that do not disturb protein binding.
In some embodiments, the binding of PTPσ to APP can be detected using immunodetection methods. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/FLAP).
The methods can be cell-based or cell-free assays.
In some embodiments, the binding between PTPσ and APP can be detected using fluorescence activated cell sorting (FACS). For example, disclosed are cell lines transfected with of PTPσ and APP fused to fluorescent proteins. These cell lines can facilitate high-throughput screens for biologically expressed and chemically synthesized molecules that disrupt the binding between PTPσ and APP.
In some embodiments, the binding between PTPσ and APP can be detected in a cell-free setting where one of these two binding partners is purified and immobilized/captured through covalent or non-covalent bond to a solid surface or beads, while the other binding partner is allowed to bind in the presence of biologically expressed and chemically synthesized molecules to screen candidate agents for their efficacies in dissociating APP-PTPσ interaction.
In some embodiments, the binding between PTPσ and APP can be detected in a setting where cell membrane preparations extracted from fresh rodent brain homogenates (containing both APP and PTPσ) are contacted with biologically expressed and chemically synthesized molecules. Subsequently, one of the binding partners is immunoprecipitated and the binding or co-immunoprecipitation of the other binding partner is detected using its specific antibody.
A candidate agent that decreases or abolishes APP-PTPσ binding in a disclosed method herein has the potential to slow, stop, reverse, or prevent neurodegeneration.
In some embodiments, the method comprising contacting/incubating a candidate compound with cell membrane preparations extracted from fresh rodent brain homogenates, wherein a decrease in APP β- and/or γ-cleavage products is an indication that the candidate agent has the potential to slow, stop, reverse, or prevent neurodegeneration. APP β- and/or γ-cleavage products can be detected by routine biochemical methods such as Western blot analysis, ELISA, and immnuopurification.
In general, candidate agents can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) used.
Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., from purveyors of chemical libraries including but not limited to ChemBridge Corporation (16981 Via Tazon, Suite G, San Diego, Calif., 92127, USA, www.chembridge.com); ChemDiv (6605 Nancy Ridge Drive, San Diego, Calif. 92121, USA); Life Chemicals (1103 Orange Center Road, Orange, Conn. 06477); Maybridge (Trevillett, Tintagel, Cornwall PL34 0HW, UK).
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including O2H, (Cambridge, UK), MerLion Pharmaceuticals Pte Ltd (Singapore Science Park II, Singapore 117528) and Galapagos NV (Generaal De Wittelaan L11 A3, B-2800 Mechelen, Belgium).
In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods or by standard synthetic methods in combination with solid phase organic synthesis, micro-wave synthesis and other rapid throughput methods known in the art to be amenable to making large numbers of compounds for screening purposes. Furthermore, if desired, any library or compound, including sample format and dissolution is readily modified and adjusted using standard chemical, physical, or biochemical methods.
Candidate agents encompass numerous chemical classes, but are most often organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons, or, in some embodiments, having a molecular weight of more than 100 and less than about 5,000 Daltons. Candidate agents can include functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, for example, at least two of the functional chemical groups. The candidate agents often contain cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
In some embodiments, the candidate agents are proteins. In some aspects, the candidate agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, can be used. In this way libraries of procaryotic and eucaryotic proteins can be made for screening using the methods herein. The libraries can be bacterial, fungal, viral, and vertebrate proteins, and human proteins.
Methods of Treatment
Disclosed herein are methods for treating neurodegenerative diseases that involve β-amyloid pathologies and/or Tau pathologies, including but not limited to Alzheimer's disease, Lewy body dementia, frontotemporal dementia, cerebral amyloid angiopathy, primary age-related tauopathy, chronic traumatic encephalopathy, Parkinson's disease, postencephalitic parkinsonism, Huntington's disease, amyolateral sclerosis, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Lytico-Bodig disease, ganglioglioma and gangliocytoma, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, and/or Creutzfeldt-Jakob disease.
These peptides, compositions, and methods may also be used to prevent these neurodegenerative diseases in populations at risk, such as people with Down syndrome and those suffered from brain injuries or cerebral ischemia, as well as the aging population.
In some embodiments, these methods involve disrupting the binding between PTPσ and APP, preventing β-amyloidogenic processing of APP without affecting other major substrates of β- and γ-secretases. For example, the methods can involve administering to a subject a peptide disclosed herein. In other embodiments, monoclonal antibodies could be formed against the IG1 domain of PTPσ or a fragment thereof, a fragment between the E1 and E2 domain of the APP695 isoform, or both, and these antibodies, or fragments thereof, could be administered to the subject.
Chondroitin sulfates (CS) and heparin or its analog heparan sulfates (HS) are two main classes of glycosaminoglycans (GAGs) in the brain that are “sensed” by neurons via Receptor Protein Tyrosine8. The ratio of CS and HS therefore affects the downstream effects of PTPσ, because CS and HS compete to interact with the receptor yet lead to opposite signaling and neuronal responses (such as neurite regeneration). CS increases but HS decreases APP β-cleavage products (Example 2). Therefore, in some embodiments, the methods involve administering to the subject a composition, which restores the physiological molecular CS/HS balance, may be used to treat and prevent aforementioned neurodegenerative diseases. These therapies could be applied alternatively or in addition to the polypeptides listed above. In some embodiments, administering HS, or its analog heparin, or their mimetics modified to reduce anti-coagulant effects, with a saccharide chain length of 17, 18, 19, 20, 21, 22, 23, 24 units or longer, could assist in restoring the physiological molecular CS/HS balance. In some embodiments, the balance is restored by administering enzymes that digest CS (such as Chondroitinase ABC) or prevent the degradation of HS (such as Heparanase inhibitors PI-88, OGT 2115, or PG545). Alternatively or in addition, agents that mimic the HS/heparin effect of PTPσ clustering8, such as multivalent antibodies, could be administered.
In some embodiments, the method involves administering a composition described herein in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1 μg to about 100 mg per kg of body weight, from about 1 μg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of composition administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Methods and Materials
Mouse lines: Mice were maintained under standard conditions approved by the Institutional Animal Care and Use Committee. Wild type and PTPσ-deficient mice of Balb/c background were provided by Dr. Michel L. Tremblay9. Homozygous TgAPP-SwDI mice, C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax, stock number 007027, were from the Jackson Laboratory. These mice express human APP transgene harboring Swedish, Dutch, and Iowa mutations, and were bred with Balb/c mice heterozygous for the PTPσ gene to generate bigenic mice heterozygous for both TgAPP-SwDI and PTPσ genes, which are hybrids of 50% C57BL/6J and 50% Balb/c genetic background. These mice were further bred with Balb/c mice heterozygous for the PTPσ gene. The offspring from this mating are used in experiments, which include littermates of the following genotypes: TgAPP-SwDI(+/−)PTPσ(+/+), mice heterozygous for TgAPP-SwDI transgene with wild type PTPα; TgAPP-SwDI(+/−)PTPσ(−/−), mice heterozygous for TgAPP-SwDI transgene with genetic depletion of PTPσ; TgAPP-SwDI(−/−) PTPσ(+/+), mice free of TgAPP-SwDI transgene with wild type PTPσ. Both TgAPP-SwDI(−/−) PTPσ(+/+) and Balb/c PTPσ(+/+) are wild type mice but with different genetic background. Heterozygous TgAPP-SwInd (J20) mice, 6.Cg-Tg(PDGFB-APPSwInd)20Lms/2Mmjax, were provided by Dr. Lennart Mucke. These mice express human APP transgene harboring Swedish and Indiana mutations, and were bred with the same strategy as described above to obtain mice with genotypes of TgAPP-SwInd (+/−)PTPσ(+/+) and TgAPP-SwInd (+/−)PTPσ(−/−).
Antibodies:
Immunohistochemistry: Adult rat and mice were perfused intracardially with fresh made 4% paraformaldehyde in cold phosphate-buffered saline (PBS). The brains were collected and post-fixed for 2 days at 4° C. Paraffin embedded sections of 10 μM thickness were collected for immunostaining. The sections were deparaffinized and sequentially rehydrated. Antigen retrieval was performed at 100° C. in Tris-EDTA buffer (pH 9.0) for 50 min. Sections were subsequently washed with distilled water and PBS, incubated at room temperature for 1 hour in blocking buffer (PBS, with 5% normal donkey serum, 5% normal goat serum, and 0.2% Triton X-100). Primary antibody incubation was performed in a humidified chamber at 4° C. overnight. After 3 washes in PBS with 0.2% Triton X-100, the sections were then incubated with a mixture of secondary and tertiary antibodies at room temperature for 2 hours. All antibodies were diluted in blocking buffer with concentrations recommended by the manufacturers. Mouse primary antibodies were detected by goat anti-mouse Alexa488 together with donkey anti-goat Alexa488 antibodies; rabbit primary antibodies were detected by chicken anti-rabbit CF568 and donkey anti-chicken Cy3 antibodies; chicken antibody was detected with donkey anti-chicken Cy3 antibody. Sections stained with only secondary and tertiary antibodies (without primary antibodies) were used as negative controls. At last, DAPI (Invitrogen, 300 nM) was applied on sections for nuclear staining. Sections were washed 5 times before mounted in Fluoromount (SouthernBiotech).
Wide field and confocal images were captured using Zeiss Axio Imager M2 and LSM780, respectively. Images are quantified using the Zen 2 Pro software and ImageJ.
Protein extraction, immunoprecipitation, and western blot analysis: For the co-immunoprecipitation of APP and PTPσ, RIPA buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate). For the co-immunoprecipitation of APP and BACE1, NP40 buffer was used (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% NP40) without or with SDS at concentration of 0.1%, 0.3%, and 0.4%. For total protein extraction and immunopurification of CTFβ, SDS concentration in RIPA buffer was adjusted to 1% to ensure protein extraction from the lipid rafts. Mouse or rat forebrains were homogenized thoroughly on ice in homogenization buffers (as mention above) containing protease and phosphatase inhibitors (Thermo Scientific). For each half of forebrain, buffer volume of at least 5 ml for mouse and 8 ml for rat was used to ensure sufficient detergent/tissue ratio. The homogenates were incubated at 4° C. for 1 hour with gentle mixing, sonicated on ice for 2 minutes in a sonic dismembrator (Fisher Scientific Model 120, with pulses of 50% output, 1 second on and 1 second off), followed with another hour of gentle mixing at 4° C. All samples were used fresh without freezing and thawing.
For co-immunoprecipitation and immunopurification, the homogenates were then centrifuged at 85,000×g for 1 hour at 4° C. and the supernatants were collected. Protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific). 0.5 mg total proteins of brain homogenates were incubated with 5 μg of designated antibody and 30 μl of Protein-A sepharose beads (50% slurry, Roche), in a total volume of 1 ml adjusted with RIPA buffer. Samples were gently mixed at 4° C. overnight. Subsequently, the beads were washed 5 times with cold immunoprecipitation buffer. Samples were then incubated in Laemmli buffer with 100 mM of DTT at 75° C. for 20 minutes and subjected to western blot analysis.
For analysis of protein expression level, the homogenates were centrifuged at 23,000×g for 30 min at 4° C. and the supernatants were collected. Protein concentration was measured using BCA Protein Assay Kit (Thermo Scientific). 30 μg of total proteins were subjected to western blot analysis.
Electrophoresis of protein samples was conducted using 4-12% Bis-Tris Bolt Plus Gels, with either MOPS or MES buffer and Novex Sharp Pre-stained Protein Standard (all from Invitrogen). Proteins were transferred to nitrocellulose membrane (0.2 μm pore size, Bio-Rad) and blotted with selected antibodies (see table above) at concentrations suggested by the manufacturers. Primary antibodies were diluted in SuperBlock TBS Blocking Buffer (Thermo Scientific) and incubated with the nitrocellulose membranes at 4° C. overnight; secondary antibodies were diluted in PBS with 5% nonfat milk and 0.2% Tween20 and incubated at room temperature for 2 hours. Membranes were washes 4 times in PBS with 0.2% Tween20 between primary and secondary antibodies and before chemiluminescent detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
Western blot band intensity was quantified by densitometry.
Aβ ELISA assays: Mouse forebrains were thoroughly homogenized in tissue homogenization buffer (2 mM Tris pH 7.4, 250 mM sucrose, 0.5 mM EDTA, 0.5 mM EGTA) containing protease inhibitor cocktail (Roche), followed by centrifugation at 135,000×g (33,500 RPM with SW50.1 rotor) for 1 hour at 4° C. Proteins in the pellets were extracted with formic acid (FA) and centrifuged at 109,000×g (30,100 RPM with SW50.1 rotor) for 1 hour at 4° C. The supernatants were collected and diluted 1:20 in neutralization buffer (1 M Tris base, 0.5 M Na2HPO4, 0.05% NaN3) and subsequently 1:3 in ELISA buffer (PBS with 0.05% Tween-20, 1% BSA, and 1 mM AEBSF). Diluted samples were loaded onto ELISA plates pre-coated with 6E10 antibody (Biolegend) to capture Aβ peptides. Serial dilutions of synthesized human Aβ 1-40 or 1-42 (American Peptide) were loaded to determine a standard curve. Aβ was detected using an HRP labeled antibody for either Aβ 1-40 or 1-42 (see table above). ELISA was developed using TMB substrate (Thermo Scientific) and reaction was stopped with 1N HCl. Plates were read at 450 nm and concentrations of Aβ in samples were determined using the standard curve.
Behavior assays: The Y-maze assay: Mice were placed in the center of the Y-maze and allowed to move freely through each arm. Their exploratory activities were recorded for 5 minutes. An arm entry is defined as when all four limbs are within the arm. For each mouse, the number of triads is counted as “spontaneous alternation”, which was then divided by the number of total arm entries, yielding a percentage score. The novel object test: On day 1, mice were exposed to empty cages (45 cm×24 cm×22 cm) with blackened walls to allow exploration and habituation to the arena. During day 2 to day 4, mice were returned to the same cage with two identical objects placed at an equal distance. On each day mice were returned to the cage at approximately the same time during the day and allowed to explore for 10 minutes. Cages and objects were cleaned with 70% ethanol between each animal. Subsequently, 2 hours after the familiarization session on day 4, mice were put back to the same cage where one of the familiar objects (randomly chosen) was replaced with a novel object, and allowed to explore for 5 minutes. Mice were scored using Observer software (Noldus) on their time duration and visiting frequency exploring either object. Object exploration was defined as facing the object and actively sniffing or touching the object, whereas any climbing behavior was not scored. The discrimination indexes reflecting interest in the novel object is denoted as either the ratio of novel object exploration to total object exploration (NO/NO+FO) or the ratio of novel object exploration to familiar object exploration (NO/FO). All tests and data analyses were conducted in a double-blinded manner.
Statistics: 2-tailed Student's t test was used for two-group comparison. Relationship between two variables was analyzed using linear regression. All error bars show standard error of the means (SEM).
Results
PTPσ is an APP Binding Partner in the Brain.
Previously identified as a neuronal receptor of extracellular proteoglycans8,10,11 PTPσ is expressed throughout the adult nervous system, most predominantly in the hippocampus12,13, one of earliest affected brain regions in AD. Using immunohistochemistry and confocal imaging, it was found that PTPσ and APP (the precursor of Aβ) colocalize in hippocampal pyramidal neurons of adult rat brains, most intensively in the initial segments of apical dendrites, and in the perinuclear and axonal regions with a punctate pattern (
Genetic Depletion of PTPσ Reduces β-Amyloidogenic Products of APP.
The molecular interaction between PTPσ and APP prompted an investigation on whether PTPσ plays a role in amyloidogenic processing of APP. In neurons, APP is mainly processed through alternative cleavage by either α- or β-secretase. These secretases release the N-terminal portion of APP from its membrane-tethering C-terminal fragment (CTFα or CTFβ, respectively), which can be further processed by the γ-secretase14,15 Sequential cleavage of APP by the β- and γ-secretases is regarded as amyloidogenic processing since it produces Aβ peptides16. When overproduced, the Aβ peptides can form soluble oligomers that trigger ramification of cytotoxic cascades, whereas progressive aggregation of Aβ eventually results in the formation of senile plaques in the brains of AD patients (
Western blot analysis with protein extracts from mouse brains showed that genetic depletion of PTPσ does not affect the expression level of full length APP (
Because CTFβ is an intermediate proteolytic product between β- and γ-cleavage, its decreased steady state level could result from either reduced production by n-cleavage or increased degradation by subsequent γ-secretase cleavage (
Curtailed Progression of β-Amyloidosis in the Absence of PTPσ.
Progressive cerebral Aβ aggregation (β-amyloidosis) is regarded as a benchmark of AD progression. To investigate the effects of PTPσ on this pathological development, Aβ deposits in the brains of 9-month old (mid-aged) and 16-month old (aged) TgAPP-SwDI mice were monitored. At age of 9 to 11 months, Aβ deposits are found predominantly in the hippocampus, especially in the hilus of the dentate gyrus (DG) (
Decreased BACE1-APP Affinity in PTPσ-Deficient Brains.
Consistent with these observations that suggest a facilitating role of PTPσ in APP β-cleavage, the data further reveal that PTPσ depletion weakens the interaction of APP with BACE1, the β-secretase in the brain. To test the in vivo affinity between BACE1 and APP, co-immunoprecipitation were performed of the enzyme and substrate from mouse brain homogenates in buffers with serially increased detergent stringency. Whereas BACE1-APP association is nearly equal in wild type and PTPσ-deficient brains under mild buffer conditions, increasing detergent stringency in the buffer unveils that the molecular complex is more vulnerable to dissociation in brains without PTPσ (
Although it cannot be ruled out that some alternative uncharacterized pathway may contribute to the parallel decrease of CTFβ and Aβ in PTPσ-deficient brains, these data consistently support the notion that PTPσ regulates APP amyloidogenic processing, likely via facilitation of BACE1 activity on APP, the initial process of Aβ production.
The Specificity of β-Amyloidogenic Regulation by PTPσ.
The constraining effect of PTPσ on APP amyloidogenic products led to further questions regarding whether this observation reflects a specific regulation of APP metabolism, or alternatively, a general modulation on the β- and γ-secretases. First, the expression level of these secretases in mouse brains were assessed with or without PTPσ. No change was found for BACE1 or the essential subunits of γ-secretase (
PTPσ Depletion Relieves Neuroinflammation and Synaptic Impairment in APP Transgenic Mice.
Substantial evidence from earlier studies has established that overproduction of Aβ in the brain elicits multiplex downstream pathological events, including chronic inflammatory responses of the glia, such as persistent astrogliosis. The reactive (inflammatory) glia would then crosstalk with neurons, evoking a vicious feedback loop that amplifies neurodegeneration during disease progression25-27.
The TgAPP-SwDI model is one of the earliest to develop neurodegenerative pathologies and behavioral deficits among many existing AD mouse models17. These mice were therefore chosen to further examine the role of PTPσ in AD pathologies downstream of neurotoxic A.
The APP-SwDI(+)PTPσ(+/+) mice, which express the TgAPP-SwDI transgene and wild type PTPσ, have developed severe neuroinflammation in the brain by the age of 9 months, as measured by the level of GFAP (glial fibrillary acidic protein), a marker of astrogliosis (
Among all brain regions, the most affected by the expression of TgAPP-SwDI transgene appears to be the hilus of the DG, where Aβ deposition and astrogliosis are both found to be the most severe (
Interestingly, the APP-SwDI(+)PTPσ(−/−) mice sometimes express higher levels of presynaptic markers in the CA3 terminal zone than their age-matched non-transgenic wild type littermates (
Tau Pathology in Aging AD Mouse Brains is Dependent on PTPσ.
Neurofibrillary tangles composed of hyperphosphorylated and aggregated Tau are commonly found in AD brains. These tangles tend to develop in a hierarchical pattern, appearing first in the entorhinal cortex before spreading to other brain regions5,6. The precise mechanism of tangle formation, however, is poorly understood. The fact that Tau tangles and Aβ deposits can be found in separate locations in postmortem brains has led to the question of whether Tau pathology in AD is independent of Aβ accumulation5,6. Additionally, despite severe cerebral β-amyloidosis in many APP transgenic mouse models, Tau tangles have not been reported, further questioning the relationship between Aβ and Tau pathologies in vivo.
Nonetheless, a few studies did show non-tangle like assemblies of Tau in dystrophic neurites surrounding Aβ plaques in APP transgenic mouse lines29-31, arguing that Aβ can be a causal factor for Tau dysregulation, despite that the precise nature of Tau pathologies may be different between human and mouse. In the histological analysis using an antibody against the proline-rich domain of Tau, Tau aggregation was observed in the brains of both TgAPP-SwDI and TgAPP-SwInd mice during the course of aging (around 9 months for the APP-SwDI(+)PTPσ(+/+) mice and 15 months for the APP-SwInd(+)PTPσ(+/+) mice) (
In both TgAPP-SwDI and TgAPP-SwInd mice, the Tau aggregates are found predominantly in the molecular layer of the piriform and entorhinal cortices, and occasionally in the hippocampal region (
Consistent with the findings in postmortem AD brains, the distribution pattern of Tau aggregates in the TgAPP-SwDI brain does not correlate with that of Aβ deposition, which is pronounced in the hippocampus yet only sporadic in the piriform or entorhinal cortex at the age of 9 months (
Next, the question of whether the expression of APP transgenes or genetic depletion of PTPσ regulates Tau aggregation by changing its expression level and/or phosphorylation status was examined. Western blot analysis of brain homogenates showed that Tau protein expression is not affected by the APP transgenes or PTPσ (
Although the underlying mechanism is still unclear, the finding of Tau pathology in these mice establishes a causal link between the expression of amyloidogenic APP transgenes and a dysregulation of Tau assembly. The data also suggest a possibility that PTPσ depletion may suppress Tau aggregation by reducing amyloidogenic products of APP.
Malfunction of Tau is broadly recognized as a neurodegenerative marker since it indicates microtubule deterioration7. The constraining effect on Tau aggregation by genetic depletion of PTPσ thus provides additional evidence for the role of this receptor as a pivotal regulator of neuronal integrity.
PTPσ Deficiency Rescues Behavioral Deficits in AD Mouse Models.
Next, the question was assessed of whether the alleviation of neuropathologies by PTPσ depletion is accompanied with a rescue from AD relevant behavioral deficits. The most common symptoms of AD include short-term memory loss and apathy among the earliest, followed by spatial disorientation amid impairment of many cognitive functions as the dementia progresses. Using Y maze and novel object assays as surrogate models, these cognitive and psychiatric features were evaluated in the TgAPP-SwDI and TgAPP-SwInd mice.
The Y-maze assay, which allows mice to freely explore three identical arms, measures their short-term spatial memory. It is based on the natural tendency of mice to alternate arm exploration without repetitions. The performance is scored by the percentage of spontaneous alternations among total arm entries, and a higher score indicates better spatial navigation. Compared to the non-transgenic wild type mice within the colony, the APP-SwDI(+)PTPσ(+/+) mice show a clear deficit in their performance. Genetic depletion of PTPσ in the APP-SwDI(+)PTPσ(−/−) mice, however, unequivocally restores the cognitive performance back to the level of non-transgenic wild type mice (
Apathy, the most common neuropsychiatric symptom reported among individuals with AD, is characterized by a loss of motivation and diminished attention to novelty, and has been increasingly adopted into early diagnosis of preclinical and early prodromal AD34-36. Many patients in early stage AD lose attention to novel aspects of their environment despite their ability to identify novel stimuli, suggesting an underlying defect in the circuitry responsible for further processing of the novel information34,35. As a key feature of apathy, such deficits in attention to novelty can be accessed by the “curiosity figures task” or the “oddball task” in patients34,35,37. These visual-based novelty encoding tasks are very similar to the novel object assay for rodents, which measures the interest of animals in a novel object (NO) when they are exposed simultaneously to a prefamiliarized object (FO). This assay was therefore used to test the attention to novelty in the APP transgenic mice. When mice are pre-trained to recognize the FO, their attention to novelty is then measured by the discrimination index denoted as the ratio of NO exploration to total object exploration (NO+FO), or alternatively, by the ratio of NO exploration to FO exploration. Whereas both ratios are commonly used, a combination of these assessments provides a more comprehensive evaluation of animal behavior. In this test, as indicated by both measurements, the expression of APP-SwDI transgene in the APP-SwDI(+)PTPσ(+/+) mice leads to a substantial decrease in NO exploration as compared to non-transgenic wild type mice (
To further verify the effects of PTPσ on these behavioral aspects, the TgAPP-SwInd mice were also tested using both assays, and similar results were observed. This confirms an improvement on both short-term spatial memory and attention to novelty upon genetic depletion of PTPσ (
Discussion
The above data showed that β-amyloidosis and several downstream disease features are dependent on PTPσ in two mouse models of genetically inherited AD. This form of AD develops inevitably in people who carry gene mutations that promote amyloidogenic processing of APP and overproduction of A. The data presented herein suggest that targeting PTPσ is a potential therapeutic approach that could overcome such dominant genetic driving forces to curtail AD progression. The advantage of this targeting strategy is that it suppresses Aβ accumulation without broadly affecting other major substrates of the β- and γ-secretases, thus predicting a more promising translational potential as compared to those in clinical trials that generically inhibit the secretases.
PTPσ was previously characterized as a neuronal receptor of the chondroitin sulfate- and heparan sulfate-proteoglycans (CSPGs and HSPGs)10,11. In response to these two classes of extracellular ligands, PTPσ functions as a “molecular switch” by regulating neuronal behavior in opposite manners8. The finding presented herein of a pivotal role for the proteoglycan sensor PTPσ in AD pathogenesis may therefore implicate an involvement of the perineuronal matrix in AD etiology.
More than 95% of AD cases are sporadic, which are not genetically inherited but likely result from insults to the brain that occurred earlier in life. AD risk factors, such as traumatic brain injury and cerebral ischemia38-41 have been shown to induce overproduction of Aβ in both human and rodents42-46 and speed up progression of this dementia in animal models47-49. However, what promotes the amyloidogenic processing of APP in these cases is still a missing piece of the puzzle in understanding the AD-causing effects of these notorious risk factors.
Coincidently, both traumatic brain injury and cerebral ischemia cause pronounced remodeling of the perineuronal microenvironment at lesion sites, marked by increased expression of CSPGs50-53, a major component of the perineuronal net that is upregulated during neuroinflammation and glial scar formation54-56. In the brains of AD patients, CSPGs were found associated with Aβ depositions, further suggesting an uncanny involvement of these proteoglycans in AD development57. On the other hand, analogues of heparan sulfate (HS, carbohydrate side chains of HSPGs that bind to PTPσ) were shown to inhibit BACE1 activity, suggesting their function in preventing Aβ overproduction58. After cerebral ischemia, however, the expression of Heparanase, an enzyme that degrades HS, was found markedly increased59. Collectively, these findings suggest a disrupted molecular balance between CSPGs and HSPGs in brains after lesion, which may ignite insidious signaling cascades preceding the onset of AD.
Further study could include investigation of a potential mechanism, whereby chronic CSPG upregulation or HSPG degradation in lesioned brains may sustain aberrant signaling through their neuronal sensor PTPσ, leading to biased processing of APP and a neurotoxic “Aβ cascade”. As such, altered signaling from PTPσ after traumatic brain injury and ischemic stroke may explain how these risk factors can trigger subsequent onset of AD. Restoring the integrity of brain microenvironment therefore could be essential in preventing AD for the population at risk.
CS and HS/heparin are two classes of PTPσ ligands in the perineuronal space that compete for binding to the same site on receptor PTPσ with similar affinities8. Increased CS/HS ratio is often found after brain injuries or ischemic stroke50-53,59, both of which are prominent risk factors for AD and alike neurodegenerative diseases.
These two classes of ligands were shown previously to oppositely regulate neuronal responses, such as neurite outgrowth, through their common receptor PTPσ. Whereas CS inhibits neurite outgrowth, HS/heparin promotes neurite outgrowth.
When tested in an in vitro assay for their effects on APP amyloidogenic processing, these PTPσ ligands again showed opposite effects. As in
Domain regions were subcloned from human APP695 (construct by Denis Selkoe and Tracy Yang labs purchased through Addgene.com) and PTPσ (constructs from Radu Aricescu lab). Recombinant APP and PTPσ proteins were tested in solid phase ELISA binding assays to define the binding regions on each partner. Neither E1 or E2 domain of APP interacts with PTPσ (data not shown), however the region in between these two APP domains (SEQ ID NO:1) appears to have high affinity with PTPσ IG1 domain (
Sequences:
Sequences for the peptides used in Example 3 are provided in Tables 3, 4, and 5.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The Lancet. Neurology 13, 319-329 (2014).
This application is a continuation of U.S. application Ser. No. 16/300,687, filed Nov. 12, 2018, which is a national stage application filed under 35 U.S.C. § 371 of PCT/US2017/032387 filed May 12, 2017, which claims the benefit of U.S. Provisional Application No. 62/335,159, filed May 12, 2016, which are hereby incorporated by reference in their entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62335159 | May 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16300687 | Nov 2018 | US |
| Child | 17482750 | US |
