Patent Application
20240197819
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This document relates to methods and materials for treating a mammal having Alzheimer's disease (AD). For example, one or more Zika virus (ZIKV) polypeptides can be administered to a mammal having, or at risk of developing, AD to treat the mammal.
Alzheimer's disease (AD), one of the top 10 leading causes of death in the United States, is a progressive disease that typically begins with mild memory loss and can lead to loss of the ability to carry on a conversation and respond to the environment affecting a person's ability to carry out daily activities (Xu et al., 2010 National vital statistics reports 58(19)). As of 2014, as many as 5 million Americans were living with AD, and the number of people with AD is expected to nearly triple to 14 million people by 2060 (Matthews et al., 201, Alzheimer's & Dementia 15:17-24). The costs of treating AD currently fall between $159 and $215 billion, and are projected to rise (Hurd et al., 2013 NEJM 368(14):1326-34).
AD is characterized by the accumulation of the β-amyloid peptide (AB) within the brains. The physiological generation of the Aβ peptide from amyloid precursor protein (APP) is the crucial step in the development of AD. APP is a transmembrane protein expressed at high levels in the brain and metabolized in a rapid and highly complex fashion.
This document provides methods and materials for treating a mammal having AD. For example, one or more ZIKV polypeptides can be administered to a mammal having, or at risk of developing, AD to treat the mammal. As demonstrated herein, APP and ZIKV can physically interact, ZIKV infection can enhance expression of APP polypeptides in both mouse brain cells as well as human mature neurons, APP polypeptides can inhibit ZIKV replication in vivo in mice, as well as in human neuronal stem cells/progenitor cells (NSC/NPC) in vitro. Also as demonstrated herein, select ZIKV polypeptides can reduce or eliminate cleavage of an APP polypeptide. Accordingly, ZIKV polypeptides can be used to treat mammals having, or at risk of developing, AD.
In general, one aspect of this document features methods for treating a mammal having AD. The methods can include, or consist essentially of, administering to a mammal having AD a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1. The mammal can be a human. The mammal can have been identified as having AD. The can be effective to reduce or eliminate a symptom of AD. The symptom of AD can be mental decline, difficulty thinking and understanding, confusion in the evening hours, delusion, disorientation, forgetfulness, making things up, mental confusion, difficulty concentrating, inability to create new memories, inability to do simple math, inability to recognize common things, aggression, agitation, difficulty with self care, irritability, meaningless repetition of own words, personality changes, restlessness, lack of restraint, wandering and getting lost, anger, apathy, general discontent, loneliness, mood swings, depression, hallucination, paranoia, inability to combine muscle movements, jumbled speech, loss of appetite, or any combinations thereof. The method can be effective to increase a level of APP polypeptides within the mammal. The polypeptide can be administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.
In another aspect, this document features methods for increasing a level of APP polypeptides within a mammal having AD. The methods can include, or consist essentially of, administering to a mammal having AD a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1. The mammal can be a human. The mammal can have been identified as having AD. The polypeptide can be administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.
In another aspect, this document features methods for reducing Aβ-amyloid plaque formation within a mammal having AD. The methods can include, or consist essentially of, administering to a mammal having AD a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1. The mammal can be a human. The mammal can have been identified as have AD. The polypeptide can be administered intraperitoneally, intravenously, intramuscularly, or subcutaneously.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
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.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fec.
This document provides methods and materials for treating a mammal having AD. For example, one or more ZIKV polypeptides (e.g., one or more ZIKV polypeptides having the ability to reduce or eliminate cleavage of an APP polypeptide) can be administered to a mammal having, or at risk of developing, AD to treat the mammal.
Any appropriate mammal (e.g., a mammal having, or at risk of developing, AD) can be treated as described herein. Examples of mammals that can have AD and can be treated as described herein (e.g., by administering one or more ZIKV polypeptides to the mammal) include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, mice, rats, sheep, goats, bears, cheetahs, and dolphins.
In some cases, methods described herein can include identifying a mammal (e.g., a human) as having AD. Any appropriate method can be used to identify a mammal as having AD. For example, physical and neurological examinations (e.g., to assess reflexes, muscle tone and strength, ability to get up from a chair and walk across the room, sense of sight and hearing, coordination, and balance), mental status testing (e.g., to test your thinking (cognitive) and memory skills, neuropsychological tests, laboratory tests (e.g., to rule out other disorders that can cause some symptoms similar to those of AD, such as a thyroid disorder or vitamin B-12 deficiency), cerebrospinal fluid (CSF) tests, electroencephalography (EEG), and/or imaging tests (e.g., brain-imaging tests such as magnetic resonance imaging (MRI), computerized tomography (CT), and/or positron emission tomography (PET) to evaluate loss (degeneration) of brain cells and/or to rule out other causes such as hemorrhages, brain tumors or strokes) can be used to identify a mammal (e.g., a human) as having AD.
When treating a mammal (e.g., a human) having, or at risk of developing, AD as described herein (e.g., by administering one or more ZIKV polypeptides to the mammal), the ZIKV polypeptide can be any ZIKV polypeptide. In some cases, a ZIKV polypeptide provided herein can reduce or eliminate cleavage of an APP polypeptide. A ZIKV polypeptide can be a full-length ZIKV polypeptide or a fragment of a ZIKV polypeptide provided that the fragment has the ability to reduce or eliminate cleavage of an APP polypeptide (e.g., a biologically active fragment). A ZIKV polypeptide can be from (e.g., can be derived from) any lineage of ZIKV. A ZIKV polypeptide can be a synthetic polypeptide. A ZIKV polypeptide can be from any clade of ZIKV. A ZIKV polypeptide can be any strain of ZIKV. Examples of ZIKVs that a ZIKV polypeptide can be derived from include, without limitation, East African ZIKV, West African ZIKV, Asian ZIKV, and South American ZIKV.
A ZIKV polypeptide provided herein (e.g., a ZIKV polypeptide that can reduce or eliminate cleavage of an APP polypeptide) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:1.
In some cases, a ZIKV polypeptide provided herein can be a substantially pure polypeptide. The term “substantially pure” as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In some cases, a substantially pure polypeptide provided herein can be a polypeptide that is synthesized to have a purity of at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent.
In some cases, a ZIKV polypeptide provided herein that consists essentially of the amino acid sequence set forth in SEQ ID NO:1 can be a polypeptide that has zero, one, or two amino acid substitutions within the articulated sequence of SEQ ID NO:1, has zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of SEQ ID NO:1, and/or has zero, one, two, three, four, or five amino acid residues following the articulated sequence of SEQ ID NO: 1, provided that the ZIKV polypeptide can reduce or eliminate cleavage of an APP polypeptide. Examples of ZIKV polypeptides that consist essentially of the amino acid sequence set forth in SEQ ID NO: 1 are set forth in Table 1.
A polypeptide provided herein (e.g., a ZIKV polypeptide that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1) can be any appropriate length. For example, a polypeptide provided herein can be from 35 amino acid residues to 55 amino acid residues (e.g., from 35 amino acid residues to 52 amino acid residues, from 35 amino acid residues to 50 amino acid residues, from 35 amino acid residues to 47 amino acid residues, from 35 amino acid residues to 45 amino acid residues, from 38 amino acid residues to 55 amino acid residues, from 40 amino acid residues to 55 amino acid residues, from 42 amino acid residues to 55 amino acid residues, from 37 amino acid residues to 53 amino acid residues, from 40 amino acid residues to 50 amino acid residues, from 38 amino acid residues to 42 amino acid residues, from 40 amino acid residues to 48 amino acid residues, or from 43 amino acid residues to 50 amino acid residues) in length.
When treating a mammal having, or at risk of developing, AD, one or more ZIKV polypeptides (e.g., one or more ZIKV polypeptides having the ability to reduce or eliminate cleavage of an APP polypeptide) can be administered to the mammal at any appropriate time. For example, one or more ZIKV polypeptides can be administered to a mammal having, or at risk of developing, AD before, during (e.g., concurrent with), and/or after the mammal exhibits one or more symptoms of AD. In some cases, one or more ZIKV polypeptides can be administered to a mammal before the mammal exhibits one or more symptoms of AD (e.g., when the mammal is asymptomatic and/or prior to AD developing). In some cases, one or more ZIKV polypeptides can be administered to a mammal after the mammal exhibits one or more symptoms of AD.
One or more ZIKV polypeptides (e.g., one or more ZIKV polypeptides having the ability to reduce or eliminate cleavage of an APP polypeptide) can be administered to a mammal in need thereof (e.g., a mammal having, or at risk of developing, AD) by any appropriate route. Administration can be local or systemic. Examples of routes of administration include, without limitation, intraperitoneal, intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, and parenteral administration.
When treating a mammal having, or at risk of developing, AD, the treatment can include the administration of a therapeutically effective amount of one or more ZIKV polypeptides (e.g., one or more ZIKV polypeptides having the ability to reduce or eliminate cleavage of an APP polypeptide). The terms “effective amount” and “therapeutically effective amount” refer to that amount of one or more ZIKV polypeptides sufficient to result in a therapeutic effect.
In some cases, treating a mammal having, or at risk of developing, AD as described herein (e.g., by administering one or more ZIKV polypeptides to the mammal) can be effective to reduce the severity of the AD and/or to reduce or eliminate one or more symptoms of the AD. In some cases, a symptom can be a cognitive symptom. In some cases, a symptom can be a behavioral symptom. In some cases, a symptom can be a mood symptom. In some cases, a symptom can be a psychological symptom. Examples of symptoms of AD can include, without limitation, mental decline, difficulty thinking and understanding, confusion in the evening hours, delusion, disorientation, forgetfulness, making things up, mental confusion, difficulty concentrating, inability to create new memories, inability to do simple math, inability to recognize common things, aggression, agitation, difficulty with self-care, irritability, meaningless repetition of own words, personality changes, restlessness, lack of restraint, wandering and getting lost, anger, apathy, general discontent, loneliness, mood swings, depression, hallucination, paranoia, inability to combine muscle movements, jumbled speech, and loss of appetite. In some cases, a symptom of AD can be as described elsewhere (see, e.g., cdc.gov/aging/aginginfo/alzheimers.htm#How; and mayoclinic.org/diseases-conditions/alzheimers-disease/symptoms-causes/syc-20350447). For example, one or more ZIKV polypeptides can be administered to a mammal in need thereof (e.g., a human having, or at risk for developing, AD) as described herein to reduce one or more symptoms of AD within the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, treating a mammal having, or at risk of developing, AD as described herein (e.g., by administering one or more ZIKV polypeptides to the mammal) can be effective to reduce or eliminate AB-amyloid plaque formation within the mammal. For example, one or more ZIKV polypeptides can be administered to a mammal in need thereof (e.g., a human having, or at risk for developing, AD) as described herein to reduce Aβ-amyloid plaque formation within the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, treating a mammal having, or at risk of developing, AD as described herein (e.g., by administering one or more ZIK V polypeptides to the mammal) can be effective to increase expression of an APP polypeptide within a mammal having, or at risk for developing, AD (e.g., resulting in an increased level of APP polypeptides within the mammal). The term “increased level” as used herein with respect to a level of an APP polypeptide in a mammal having, or at risk for developing, AD refers to any level that is greater than the level of that APP polypeptide observed in that mammal prior to being treated as described herein. In some cases, an increased level of an APP polypeptide can be a level that is at least 5 percent (e.g., at least 10, at least 15, at least 20, at least 25, at least 35, at least 50, at least 75, at least 100, or at least 150 percent) higher than the level of that APP polypeptide prior to being treated as described herein. In some cases, when samples have an undetectable level of an APP polypeptide prior to treatment as described herein, an increased level can be any detectable level of an APP polypeptide. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an increased level. For example, one or more ZIKV polypeptides can be administered to a mammal in need thereof (e.g., a human having, or at risk for developing, AD) as described herein to reduce increase expression of an APP polypeptide within the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Any appropriate method can be used to determine whether or not a level of APP polypeptides has been increased. For example, quantitative RT-PCR (RT-qPCR), western blotting, and/or ELISAs can be used to determine whether or not level of APP polypeptides has been increased.
In some cases, one or more ZIKV polypeptides can be administered to a mammal having, or at risk of developing, AD in the absence of any carriers (e.g., additives, fillers, vehicles, and/or diluents).
In some cases, one or more ZIKV polypeptides can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having, or at risk of developing, AD. For example, one or more ZIKV polypeptides can be formulated together with one or more pharmaceutically acceptable carriers (e.g., additives, fillers, vehicles, and/or diluents). In some cases, pharmaceutically acceptable carrier can be non-naturally occurring. Pharmaceutically acceptable carriers that can be used in a pharmaceutical composition described herein include, without limitation, dextrose, methanol, dimethyl sulfoxide (DMSO), ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat.
In some cases, a composition including one or more ZIKV polypeptides to be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal having, or at risk of developing, AD) can include one or more ZIKV polypeptides as the sole active ingredient used to treat AD.
In some cases, a composition including one or more ZIKV polypeptides to be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal having, or at risk of developing, AD) can include one or more ZIKV polypeptides together with one or more additional active ingredients (e.g., active ingredients that can be used to treat a mammal having, or at risk of developing, AD). Examples of additional active ingredients that can be used to treat a mammal having, or at risk of developing, AD include, without limitation, cholinesterase inhibitors (e.g., donepezil, galantamine, and rivastigmine), and memantine.
A composition including one or more ZIKV polypeptides can be designed for any route of administration. For example, a composition including one or more ZIKV polypeptides can be designed for parenteral (e.g., intraperitoneal) administration. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. For example, a composition including one or more ZIKV polypeptides can be designed for oral administration. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules.
A composition including one or more ZIKV polypeptides can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal having, or at risk of developing, AD) in any appropriate amount (e.g., any appropriate dose). An effective amount of a composition including one or more ZIKV polypeptides can be any amount that reduces the severity and/or reduces or eliminates one or more symptoms of AD without producing significant toxicity to the mammal. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and level of severity of the AD may require an increase or decrease in the actual effective amount administered.
A composition including one or more ZIKV polypeptides can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal having, or at risk of developing, AD) in any appropriate frequency. The frequency of administration can be any frequency that reduces the severity of the AD and/or reduces or eliminates one or more symptoms of the AD without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about ten times a day, from about three times a day to about eight times a day, or from about four times a day to about six times a day. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and level of severity of the AD may require an increase or decrease in administration frequency.
A composition including one or more ZIKV polypeptides can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal having, or at risk of developing, AD) for any appropriate duration. An effective duration for administering a composition including one or more ZIKV polypeptides can be any duration that reduces the severity of the AD and/or reduces or eliminates one or more symptoms of the AD without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several months or years to a lifetime. In some cases, the effective duration for the treatment of mammal in need thereof can range in duration from about 2 days to about a week. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and level of severity of the AD.
In some cases, methods described herein also can include administering to a mammal in need thereof (e.g., a mammal having, or at risk of developing, AD) one or more additional treatments used to treat a mammal having, or at risk of developing, AD. Examples of additional active ingredients that can be used to treat a mammal having, or at risk of developing, AD include, without limitation, cholinesterase inhibitors (e.g., donepezil, galantamine, and rivastigmine), and memantine. In cases where a mammal having, or at risk of developing, AD is treated with one or more non-nucleoside ZIKV polypeptides and is treated with one or more additional agents used to treat AD, the additional treatment used to treat AD can be administered at the same time or independently. For example, when administered independently, the one or more ZIKV polypeptides can be administered first, and the one or more additional treatment used to treat AD can be administered second, or vice versa.
In certain instances, a course of treatment and the severity of one or more symptoms related to the condition being treated (e.g., AD) can be monitored. Any appropriate method can be used to determine whether or not the severity of one or more symptoms is reduced or eliminated. For example, the severity of a ZIKV infection can be assessed using any appropriate methods and/or techniques and can be assessed at different time points. For example, physical and neurological examinations (e.g., to assess reflexes, muscle tone and strength, ability to get up from a chair and walk across the room, sense of sight and hearing, coordination, and balance), mental status testing (e.g., to test your thinking (cognitive) and memory skills, neuropsychological tests, laboratory tests (e.g., to rule out other disorders that can cause some symptoms similar to those of AD, such as a thyroid disorder or vitamin B-12 deficiency), CSF tests, EEG, and/or imaging tests (e.g., brain-imaging tests such as MRI, CT, and/or PET to evaluate loss (degeneration) of brain cells and/or to rule out other causes such as hemorrhages, brain tumors or strokes) can be used to determine the severity of one or more symptoms of a ZIKV infection.
In some cases, one or more ZIKV polypeptides can be used to treat a mammal having a disease or disorder associated with increased levels of Aβ polypeptides and/or decreased levels of APP polypeptides. Examples of diseases and disorders associated with increased levels of Aβ polypeptides and/or decreased levels of APP polypeptides include, without limitation, cerebral amyloid angiopathy.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
ZIKV Interacts with APP Protein
Using information about the ZIKV virion structure, we applied protein structure alignment methods, such as SSM and TM-alignment, to screen structurally homologous proteins with known binding partners to ZIKV virion and E protein. The binding partners were considered as potential ZIKV binding candidates. Dendritic Cell-Specific Intercellular adhesionmolecule-3-Grabbing Non-integrin (DC-SIGN) (also known as cluster of differentiation 209) is one of the cellular receptors for ZIKV. This method predicted that ZIKV E protein interacted with members of the C-type lectin receptor family 4, including DC-SIGN (data not shown).
Beta-secretase 1 (BACE1) was a hit of the screening, and there were some structural similarities between BACE1 and ZIKV virion, particularly within the ZIKV E protein. BACE1 is a transmembrane protein that binds to and cleaves APP proteins. PDB IDs for structures of ZIKV E and BACE1, obtained from the PDB Bank (rcsb.org/), are 5IRE chain A and 3HW1 chain A, respectively. Alignments of the ZIKV E and BACE1 had shown only limited structural similarities between the two proteins: both proteins have a similar β-hairpin structure, a 2-stranded β-sheets (
To examine whether APP was related to ZIKV infection, human embryonic kidney fibroblast 293 cells (HEK293) were used for two reasons: 1) the cells are not susceptible for ZIKV infection; and 2) the cells have very low levels of endogenous APP. First, we tested whether APP was a factor for ZIKV attachment to cells. N-terminal FLAG-tagged full-length APP expression plasmids were transfected into the HEK293 cells, and one day later, the transfected cells were mixed with ZIKV for one hour. The cells were washed extensively to remove the unbound viruses. The attachments of ZIKV to transfected cells were examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
ZIKV bound to APP-expressing cells more strongly than the control vector-transfected HEK293 cells (
BBS1 antibody targets the BACE1 cleavage site of APP. Because ZIKV may bind to a similar site based on molecular structural analyses (
Adult brains seem to be more resistant to ZIKV infection. One possible explanation is that adult brains have very few or no NPCs/NSCs. We suspected that adult brain cells, with the expression of APP, might trap ZIKV and thus protected NPCs/NSCs or other infectable cells from ZIKV infection. To test the hypothesis, cortical cortexes from aging (>1-year-old) APP-null (B6.129S7-Apptm1Dbo/J) and age-matched control wild type mice (WT, C57BL/6J) were isolated and passed through cell strainers (see Methods for details). The same amounts of cell clumps were dispensed and immediately incubated with ZIKV for one hour, and the amounts of free viruses remaining in the media were determined as a measure of viral absorptions to brain cortexes. Culture media from explant APP-null brain cortexes had higher levels of ZIKV viral genomes than those in WT mice, suggesting that ZIKV was being trapped by APP protein (
ZIKV E protein is considered as the major virion protein for attachment to host cells. Interestingly, PrM-E complex has been shown to be a good candidate for ZIKV vaccine, which suggested that PrM may help E protein to generate the correct conformation for interaction with host proteins. To test the potential interaction of ZIKV-APP further, we employed co-immunoprecipitation assay (co-IP) for potential ZIKV and APP interactions. By co-transfection of PrM-E and APP expression plasmids in HEK293 cells, a ZIKV-E antibody immunoprecipitated APP proteins in transfected cell lysates (
To confirm the co-IP results, whether ZIKV E and APP proteins were co-localized in the same cells was examined. As shown in
APP is quickly metabolized through an orderly process by multiple proteases. Whether ZIKV binding affected APP expression was examined. HEK293 cells were transfected with APP expressing plasmids and the cells were incubated with ZIKV overnight. APP-expressing cells exposed to ZIKV exhibited elevated levels of the APP protein than those non-exposed cells (
We suspected that the loop in the specific structure of ZIKV E protein was responsible for the potential interaction with APP (
Although ZIKV could bind to APP-expressing cells (
The recombinant lentiviruses expressing APP siRNA were used to infect NPCs/NSCs. The APP-knockdown cells were then infected by ZIKV. The viral replications were monitored by the qRT-PCR two days later. The culture supernatants from APP-knockdown cells had higher viral genomic RNA copies than those from control cells (
The scFv of BBS1 antibody expressing phages could inhibit ZIKV-interactions with APP (
Due to the fact that brains have different NPSs/NSCs proportions in different developmental stages, both neonatal and adult mice were examined for their susceptibility to ZIKV infections. The postnatal day 4 (P4) neonates were injected intracranially (i.c.) with ZIKV (1.5×10{circumflex over ( )}4 pfu), and the differences in survival rates between APP-null and WT mice were compared (
In P7 neonates, however, the dosage of ZIKV (1.5×10{circumflex over ( )}4 pfu) that caused serious neonate death in P4 (
All those data suggest that APP is a negative regulator of ZIKV replication in vitro in human NPCs/NSCs, and in vivo in mouse brains. The inhibitory effects of APP on ZIKV replication seems to be associated with the ages of mice.
C57BL/6J and APP-null mice (app−/−; B6.129S7-Apptm1Dbo/J) mice were purchased from Jackson Laboratories. All mice were bred and housed at the American Association for Accreditation of Laboratory Animal Care-accredited facility under specific-pathogen-free conditions. All experimental protocols were approved by the Institutional Animal Care and Use Committee and followed federal guidelines.
For cortical cortex culture, aged mice (>1 year old) were euthanized with CO2. The cerebrum was removed after cutting the cranium from the neck to the nose. A midline incision between the hemispheres were performed, and the cortex from the brain were peeled, cut into small pieces (1-2 mm3), and passed through 70 μm cell strainers first, then a 40 μm one subsequently. The cell clumps were washed twice with DMEM plus 10% fetal bovine serum (FBS), counted, and dispensed at desired concentrations. The cells clumps were immediately incubated with ZIKV at 37° C. for one hour for viral absorption study. For whole brain explant culture, cerebrums were isolated and cut into smaller pieces and passed through 70 μm and 40 μm cell strainers. The cell clumps were used directly for ZIKV infections.
Four day old (P4), one week old (P7), and four week old (P28) mice were intracranially (i.c.) injected with 15 μl solutions with 31 G syringes for P4 and P7 mice or 50 μl solutions with 29 G syringes for P28 mice. Hypodermic anesthesia (P4, P7 mice) or isoflurane anesthesia (P28 mice) were used following standard protocol. Animals were observed daily for clinical illness. The brains were collected at 2 day post inoculation (dpi) for P4 and P7 mice, and 4 dpi for P28 mice. RNA were isolated and used for measurement of ZIKV RNA. Mortality was observed for a period of four days.
Human embryonic kidney fibroblast 293 (or HEK 293, CRL-1573) and its derivative 293T cells, African green monkey kidney cell line Vero, Human hepatoma cell lines Huh7.5 (gifts from Dr. Padmanabhan), human neuroblastoma cells SH-SY5Y were obtained from the ATCC. The cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin (PS) in a humidified chamber with 5% CO2 at 37° C.
The Zika virus strain PRVABC59 were obtained from the Centers for Disease Control and Prevention, Fort Collins, Colorado, USA. The virus were prepared in Vero cells or Huh7.5 cells. The stock virus was stored in small aliquots at −80° C. Virus titers were determined by plaque assay on Vero cells. Basically, duplicates of serial 10-fold dilutions of virus were applied to Vero cell monolayers in 24-well plates.
The inoculum was removed after one hour incubation, and the cell monolayers were overlaid with medium containing 1% low-gelling-temperature agarose. The cells were fixed in 10% formaldehyde in PBS for 30 min, after 5 days incubation. The agarose plugs were removed, and the cells were stained with 0.1% crystal violet in 30% methanol. Plaques were counted and virus titers were calculated. The pooled human APP siRNA/shRNA/RNAi Lentivirus (siAPP; cat #: iV001195) and its control, Scrambled siRNA GFP Lentivirus (siScramble; cat #: LVP015-G) were both purchased from Applied Biological Materials Inc.
The pCAX FLAG APP (Addgene plasmid #30154) is a FLAG-tagged APP expression plasmid APP and pCAX APP 695 (Addgene plasmid #30137), an expression plasmid for predominant form of APP in the brains. pCI-neo-ZIKV-prME is expression plasmid for PrM-E protein. pcDNA3 (Invitrogen) is a cloning vector used as controls for transfections.
The antibodies for β-amyloid (B-4) (sc-28365), glyceraldehyde-3-phosphate dehydrogenase (0411) (sc-47724), and goat anti-mouse IgG-HRP (sc-2005) were from Santa Cruz. APP C-terminal Ab (Clone C1/6.1; Cat #: 802803) and ZIKV E antibody (GTX133314) were from Biolegend and GeneTex respectively. Donkey anti-Rabbit IgG Secondary Antibody Alexa Fluor 488 conjugate; Cat #A-21206 and Donkey anti-Mouse IgG Secondary Antibody Alexa Fluor 647 conjugate; Cat #A-31571 were both purchased form ThermoFisher Scientific. Tubulin antibody (Cat #: T6557) was purchased from Sigma.
Human Pluripotent Stem Cells (hPSCs) Differentiation into Neural Progenitor Cells (hNPCs) and Mature Cortical Neurons and ZIKV Infection
The hPSCs (H9 hESCs) were purchased from WiCell Research Institute (Cat #WA09, WiCell). The hPSCs were maintained in 6-well plate coated with Matrigel (Cat #354277, BD Biosciences) in Essential 8™ medium (E8, Cat #A1517001, Invitrogen). Cells were passaged every 4 days with 0.5 mM EDTA (Cat #AM9260G, Invitrogen). Media were changed daily. Cells were routinely checked for the expression of pluripotency markers, OCT4 (Cat #962649, R&D System) and NANOG (Cat #963488, R&D System), their capability to form teratomas in immunodeficient mice, their karyotypes, bacterial and mycoplasma contaminations. The hPSCs were dissociated with accutase (Cat #A1110501, Life Technologies) and plated in Matrigel-coated 6 well plates (2×106 cells/well) and cultured in E8 medium overnight to reach >90% confluency. E8 medium was removed and replaced with neural induction medium consisting of Essential 6™ medium (E6, Cat #A1516401, Invitrogen) supplied with 100 nM LDN193189 (Cat #S2618, Selleckchem) and 10 μM SB431542 (Cat #S1067, Selleckchem) for 11 days. The resulting cells were considered as hNPCs, and their identities were confirmed. The hNPCs were infected with a pooled human APP siRNA/shRNA/RNAi Lentivirus (siAPP; cat #: iV001195) and its control, Scrambled siRNA GFP Lentivirus (siScramble; cat #: LVP015-G; both from Applied Biological Materials Inc.) at 1 MOI overnight, and second day, cells were washed and infected with ZIKV (1 MOI) and one hour later, the media were replaced with fresh media. Two days later, cell media were collected and subjected to real time qRT-PCR assays. For BBS1Ab phage treatments, hNPCs were incubated with ZIKV (1 MOI) in the presence with either BBS1Ab expressing or helper phages (1 transforming unit per cell) for one hour at 37° C. Cells were washed with fresh media the uninfected viruses were removed. Two days later, cell media were subjected to qRT-PCR analysis.
To differentiate hNPCs into the cortical neurons, hNPCs were harvested on day 11 and re-plated to Matrigel-coated 6 well plates, and cultured in neural differentiation medium consisting of Neurobasal® Media (Cat #21103049, Life Technologies), B27 (50×, Cat #17504044, Life Technologies), BDNF (20 ng/ml, Cat #450-02, PeproTech), GDNF (10 ng/ml, Cat #450-10, PeproTech), L-ascorbic acid (200 μM, Cat #NC0602549, Life Technologies), DAPT (2.5 μM, Cat #S2215, Selleckchem), Dibutyryl-CAMP (0.5 mM, Cat #sc-201567A, Santa Cruz Biotechnology) for another 19 days. Half medium was changed every two days. The identities of the mature neurons were Tbr1+(Cat #ab31940, Abcam) and Tuj1+(Cat #T8578, Sigma) dual positive. The cells were infected with ZIKV and treated with CHX as described in the text and Figure legends.
Total RNA was extracted from BBS2 hybridoma cells using Tri-reagent (#93289 Sigma-Aldrich). The cDNA was synthesized from the total RNA by M-mulV reverse transcriptase (11062603001—Sigma-Aldrich) using oligodT23 primer (O4387—Sigma-Aldrich). The variable domains of the murine light and heavy chains were amplified and sequenced. Based on this, sequence specific primers for BBS2 Vh and Vl were designed harboring the appropriate restriction sites. Next BBS Vh and Vl were cloned into PCC 16 phagemid. First the Vh was cloned using NcoI (#10835315001 Sigma-Aldrich) and BclI restriction (#10693952001 Sigma-Aldrich) sites, followed by Vl cloning based on EcoRI (#10200310001 Sigma-Aldrich) and NotI (#11014714001 Sigma-Aldrich) restriction sites, yielding PCC16-BBS ScFv phagemid. For production, TG1 bacteria (#LUC60502-2 Sigma-Aldrich) containing the PCC16-BBS ScFv phagemid were grown at 37° C. in 10 ml of 2YT medium (#Y2377 Sigma-Aldrich) with 100 μg/ml Amp (#40345717748211 Sigma-Aldrich) up to OD600˜0.5, infected with 1:100 M13K07 helper phage (#N0315S-NEB) and incubated for 30 min without shaking followed by 30 min with 100 rpm shaking at 37° C. The infected cells were then grown in IL of 2YT with 100 μg/ml Amp and 70 μg/ml Kan (#60615 Sigma-Aldrich) O/N at 30° C. with 250 rpm shaking. The next day the growing media (containing the phages) was collected and phages were purified by PEG/NaCl (#1546605, S7653 Sigma Aldrich) precipitation twice followed by Cs gradient purification. The biding capacity of the phages was evaluated using ELISA. Phage-BBs ScFv was able to bind MAP peptide representing the BACE cleavage site on APP with high affinity compared to almost no background of Helper phage.
Attractene Transfection Reagent (Cat #: 301007; Qiagen) was used for transfection of 293 or 293T cells. One day later, ZIKV were incubated with the cells for one hour at 37° C. The cells were washed three times with 1×PBS and used for RNA isolation for RT-PCR analyses. For explant cultures, different amounts of viruses were incubated with cortical cortex explant cultures ate 37ºC for one hour. The culture media were centrifuged in an Eppendorf centrifuge at maximum speed for one minute and the supernatants were used to isolate RNA using QIAamp Viral RNA Mini Kit (Cat #: 52904, Qiagen). The equal amounts of RNA solutions
293 cells were transfected with PrM-E and APP695 expression plasmids at 1:1 ratios. Next day the transfected cells were washed once with 1×PBS, fixed with 4% paraformaldehyde (Sigma) for 15 min at room temperature. Cells were permeabilized and blocked with 0.1% Triton X-100 (Sigma) and 0.4% bovine serum albumin (BSA; A7906; Sigma) in PBS for 20 minutes. Samples were then incubated with APP C-terminal Ab (1:200 dilutions) and ZIKV E antibody (1:800 dilutions) for one hour, followed by incubation with secondary antibodies (Donkey anti-Rabbit IgG Secondary Antibody Alexa Fluor 488 conjugate and Donkey anti-Mouse IgG Secondary Antibody Alexa Fluor 647 conjugate) for one more hour at room temperature. Both secondary antibodies were used with 1:1000 dilutions and the dilution buffer for primary and secondary antibodies was 0.1% Tween 20 (Sigma) and 0.4% bovine serum albumin (BSA; A7906; Sigma) in 1×PBS. The samples were then stained with DAPI (4′,6-diamidino-2-phenylindole), washed three times with PBS and mounded. All samples were examined a Nikon-Ti2 fluorescence microscope and images were obtained using a Nikon A1r-Ti2 confocal system at the Microscopy Core Facility at the UNL.
Western Blot Analysis with Enhanced Chemiluminescence (ECL)
Separation of proteins on SDS-PAGE was carried out following the standard protocol. After the proteins were transferred to a nitrocellulose or Immobilon membrane, the membrane was blocked with 5% nonfat dry milk in TBST (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.05% Tween-20) at room temperature for 30 minutes. It was washed briefly with TBST, and incubated with the primary antibody in 5% milk in TBST for 1 hour at room temperature, or overnight at 4° C. After washing the membrane with TBST three times (10 minutes each), it was incubated with the secondary antibody at room temperature for 1 h. The membrane was then washed three times with TBST, treated with ECL detection reagents, and exposed to BlueBlot™ HS film from Life Science Products (XR-0810-100). The intensities of the target signals were measured by a BioRad ChemiDoc MP Imaging system.
Quantitative Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
For RNA from brains or cells, TRIzol™ Reagents (Cat #: 15596026; Invitrogen) were used for RNA isolation with standard protocol. For viral RNA in the media, QIAamp Viral RNA Mini Kits (Cat #: 52904; Qiagen) were used following manufacturer's recommendations. SuperScript™ II Reverse Transcriptase (Cat #: 18064014; Invitrogen) were used for first cDNA strand synthesis. Routine methods for semi-quantitative RT-PCR were performed as described. The ZIKV primers were: ZIK-F: 5′-CCG CTG CCC AAC ACA AG-3′ (SEQ ID NO:11); ZIK-R: 5′-CCA CTA ACG TTC TTT TGC AGA CAT-3′ (SEQ ID NO:12). The human actin primers were: Actin1: 5′-TTC TAC AAT GAG CTG CGT GT-3′ (SEQ ID NO:13), and Actin 2: 5′-GCC AGA CAG CAC TGT GTT GG-3′ (SEQ ID NO:14). For qRT-PCR, abundance of target RNAs was quantified by CFX96 Real-Time System (BIORAD) or Applied Biosystems Step One plus Real Time PCR system following the manufacturers' recommendations. The ZIKV primers were ZIKA-F and -R. Primers for mouse GAPDH: 5′-GAA GGT GAA GGT CGG AGT A-3 (SEQ ID NO:15)′ and 5′-GAA GAT GGT GAT GGG ATT TC-3′ (SEQ ID NO:16).
The probes for ZIKV and GAPDH were 5′-AGC CTA CCT TGA CAA GCA ATC AGA CAC TCA A-3 (SEQ ID NO:17)′ and 5′-CAA GCT TCC CGT TCT CAG CC-3′ (SEQ ID NO:18) respectively. The probes were labelled with 6-carboxyfluorescein phosphoramidite (FAM) reporter dye at the 5′ end and 6-carboxytetramethylrhodamine (TAMRA) at the 3′ end. The 2{circumflex over ( )}-ΔΔCt method was used for calculation of the relative ZIKV expression (ZIKV/GAPDH).
For transfection of 293 cells, the Attractene Transfection Reagent (Cat #: 301007; Qiagen) was used following manufacturers recommendations. Protein biosynthesis inhibitor, cycloheximide (CHX), was purchased from Sigma (C7698). The cells were treated with CHX (100 μg/ml) with ZIKV simultaneously and cell lysates were made at indicated times and used for Western blot analysis. For mature neurons, the cells were incubated with ZIKV for one hour, and then CHX was added, and cell lysates were collected at indicated times.
General statistical analyses were performed by functions implemented in Microsoft Excel and R. Two group comparisons were done with Student's two-tailed unpaired T-test. The R package of “Survival” was used to fit and plot the survival curves.
Amyloid precursor protein (APP) is a membrane protein predominantly expressed in brains and metabolized in a rapid and highly complex fashion by a series of proteases (
ZIKV cannot infect immunocompetent mice efficiently, but STAT2-null mice are susceptible to ZIKV. We tested weather APP was modulated by ZIKV in other organs in STAT2-null mice. Mice were infected with ZIKV for various times and euthanized for tissue collections. APP protein levels were increased in splenocytes as early as Day 2 post infections (dpi 2). However, APP expression in the lung were not increased at dpi2 but increased at dpi 4 and 5 (
ZIKV NS5 promotes proteasome degradation of human signal transducer and activator of transcription 2 (STAT2). Because STAT2 is a critical mediator of Type I IFN signaling, ZIKV limits the IFN response during human infection. However, ZIKV NS5 does not destroy mouse STAT2, allowing an efficient and effective IFN response. ZIKV cannot infect immunocompetent mice efficiently, but STAT2-null mice are susceptible to ZIKV. Therefore, the role of APP in ZIKV-mediated pathogenesis may be best illustrated in STAT2-null background that represents a similar situation to ZIKV infections in humans.
APP-null and STAT2-null mice, both on C57BL/6 background, were crossed for double knockout genotype (DKO; app−/−; stat2−/−). We first confirmed DKO mice with genotyping experiments (data not shown), and further with protein expression data (
As demonstrated in Example 1 and Example 2, 1) APP is apparently another binding target of ZIKV (
How does ZIKV Increase the Expression of APP In Vivo?
Hypothesis: A ZIKV-induced factor(s) is responsible for the systematic induction of APP
Rationale and Significance: We have found that ZIKV can stabilize APP protein (
The induction of APP proteins apparently can last from dpi 2-8 in the spleen. As spleen is one of the major sites for ZIKV replication, we concentrate on splenocytes and choose two early time points (dpi 2 and dpi 4) for analysis the potential changes that are associated with APP expression (
Cytokine profiles in plasma. Plasma in triplicates are used for cytokine detection using multiplexing technology. We use the Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay (BioRad), which allows simultaneous assay for 23 mouse cytokines for each sample including IFNγ, IL-1, -6, and TNFα. IL-1β, IL-6, IFNγ, and TNFα are highly expressed upon ZIKV infection at both time points.
Gene expression profiles in splenocytes. Splenocytes are isolated from mice and separated into two parts: one for RNA isolation, another for FACS analyses. RNAs are extracted and subjected for a next-generation RNA sequencing (RNA-seq) analysis. The sixteen samples are multiplexed and distributed on a single NextSeq HighOutput Flowcell V2 75 cycles to generate approximately 16 to 20 million 75 bp single reads per sample. After the initial quality control step to remove low quality reads or nucleotides, all processed RNA-Seq reads are mapped onto the reference human genomes using Bowtie with up to two base mismatches allowed per read. Numbers of reads in genes are counted by the HTSeq counting tool with gene annotations. For pair-wise comparisons, the edgeR package with TMM normalization method is used to analyze the numbers of reads aligned to genes and to identify differentially expressed genes. A threshold value for fold-change of differential expression is set at Fold Change>2, and the adjusted P-values<0.001 are used to reject the null hypothesis. By comparing gene expression profiles, specific and common genes targeted by ZIKV are identified. We perform additional analysis for functional classification, gene ontology term enrichment test, canonical pathway analyses, Ingenuity Pathway Analyses; and database and literature mining. We obtain a whole picture of the RNA targets and signaling pathway. We identify: 1) top four pathways that are the most significantly regulated by ZIKV; 2) cell type changes based on specific lineage marks; 3) major ZIKV targets; and finally 4) APP mRNA levels after ZIKV infection.
Determination which cell population(s) expresses APP. First, we use several commercially available APP antibodies to conjugate with PE and identify proper antibodies and conditions for analyses of APP-expressing cells in splenocytes from ZIKV infected mice. Once the condition is established, we combine with other antibodies with proper labels for analyses of variety of specific cells. Our FACS Core Facility have capacity to analyses 16 different lasers simultaneously. The proportion of cells, and the levels of APP expression are obtained. Many kinds of cells play a role in anti-ZIKV responses. We initially check the proportion of the CD4 and CD8 T lymphocytes, Treg (CD25), B lymphocytes, NK cells, macrophages, dendritic cells with their specific markers. We combine all the data together and analyze. All information would lead to clues for a putative factor(s) that might lead to high APP expression.
Once we have the putative targets for induction of APP in vivo, we first use the factor(s) to treat STAT2-null mice. Basically, nine mice are used (3 for PBS, 6 for cytokine combinations with various concentrations). Putative cytokine(s) in combinations are used to treat the target mice and two days later, the mice are euthanized and splenocytes are examined by FACS and Western Blot for APP expressions. The cytokines are chosen based on the signaling pathways activated by ZIKV infection and cells for APP expression. Second, if blocking agents or antagonists are available, such as IL6 antibody, we test whether blocking the cytokine would affect ZIKV-mediated APP expression. ZIKV is used to infect STAT2-null mice and at the same time, single or multiple antagonists are injected. Whether those antagonists could alter the induction of APP is examined two days later as in
Outcomes: A cytokine(s) and a cell type(s) is identified to be associated with APP induction in vivo. It is possible that several cytokines together may induce APP expressions in vivo. Currently, IL-6 and IL-1B are the two major candidates for APP mRNA inductions. However, if APP mRNA is not changed accordingly, IL-1B is the primary candidate as it enhances APP mRNA translation efficiency. These experiments may establish that APP may not just a restriction factor for the host brain, but also an inducible factor against ZIKV in innate immunity.
Alternative Strategies: A. Plasma ApoE levels are measured after ZIKV infection because ApoE induces APP mRNA expression in human neurons. If ApoE is increased upon ZIKV infections, we test if ApoE treatment could increase APP in splenocytes in vitro and in vivo. B. If in vivo induction by cytokines are not working properly, we inject cytokines plus inactivated ZIKV. Maybe the cytokines plus ZIKV virions might have some unexpected outcomes. C. In vitro induction assay is used. Basically, the target primary cells are sorted out with flow cytometry from an uninfected mouse. The cytokine(s), with or without ZIKV, are used for ZIKV infection and the cells are examined by FACS or Western Blot for APP expressions two days later. D. The test for in vivo induction with antagonist might be a challenge. A combination of multiple antagonists might be necessity. If Alternative C is working, we test the antagonists in combination in an in vitro setting first, before moving to in vivo experiments. E. No sex differences are expected.
How does a Viral Peptide Enhance APP Expression?
Hypothesis: ZIKV peptide binds to APP directly to disrupt APP proteolytic processing.
Rationale and Significance: A peptide is identified to stabilize APP expression (
Determine Whether the Viral Peptide Interact with APP Directly
The peptide (P2) could enhance the expression of APP, apparently through the stabilization of APP protein (
First, we generate a P2-based peptide antibody. A P2SA peptide, GMI VND TGH ETD ENR AKV EI (SEQ ID NO:19), 20-aa stretch from the P2 sequence, is synthesized with an addition of cysteine in the N-terminus and sent out for antibody production. The peptide sequences were analyzed and found to be good for generating antibodies based on available programs. We test if rabbit sera are reacting with P2 properly. If the peptide antibody was not working properly, we synthesize two new P2 peptides with FLAG tag linked to N- and C-terminus respectively. Whether the FLAG-P2s enhance APP expression is examined. If they work, we use the FLAG-P2 for experiments below with FLAG antibody instead. Second, we transfect P2 peptide with APP-expression plasmid into HEK293 cells and examine their potential interactions. We do: 1) Co-immunoprecipitation (Co-IP) experiments. APP and P2 peptide antibodies are used for co-IP experiment as shown in
We test whether the P2 peptide could interact with one of the processed APP product. The sAPPα has majority of APP's ectodomain and contains the predicted ZIKV-binding site (
Whether P2 peptide affects APP processing in transfected cells is examined. Basically, we transfect APP expression plasmid into cells, with or without P2 peptide. The expression of APP processed products, amyloid beta 42 (Aβ42), Aβ40, soluble Aβ precursor protein alpha (sAPPα), sAPPβ and APP intracellular domain (AICD), is examined in both media and cell lysates. The routine ELISA and Western blot analyses are employed depending on the targets. Reagents are selected carefully as some precautionary notes present. The difference in APP processing with or without P2 peptide, both in quality as well as quantities are obtained. The critical cleavage site(s), influenced by ZIKV binding, is/are determined based on cleavage products. In addition, some new peptide(s) or cleavage site(s) may be generated. The new peptides are further examined by The Proteomics Core Facility at UNL.
Whether P2-APP interaction affects APP processing and stability is tested. We: A) compare the behavior of P2 and P2SA peptide. P2SA peptide (used for antibody production) does not interact with APP. We compare the two peptides in their ability to interact with APP and their capacity to increase APP expression. B) make a deletional mutagenesis in APP expression plasmids and remove the potential interacting site with ZIKV and P2. Whether the mutant APP protein has a defect in its processing, interaction with and enhancement by P2 peptide is examined in HEK293 cells. The result implicates the role of P2-APP interaction in APP processing and enhancement.
Other than protease processing, whether proteasome pathway plays a role in ZIKV-mediated APP stabilization in vitro is be determined. APP and its processed product accumulations are affected by ubiquitination. It is possible that ZIKV affects proteasome pathway and in turn stabilizes APP proteins. We make a mutagenesis in APP expression plasmids and change three lysine residues into arginines (aa #724-726; from NM_201414.2). It has been reported that the mutant (APP3R) has drastically reduced the ubiquitination levels of APP at least in Hela cells. Whether the APP3R mutant protein is enhanced by the ZIKV infection is tested in HeLa as well as HEK293 cells. The result implicates the role of ubiquitination in ZIKV-mediated enhancement of APP protein. Second, the proteasome inhibitors, lactacystin and M132, are used to treat cells followed by ZIKV infection, and APP expression is then examined. Whether reduction of ubiquitin (via siRNA targets Ub) and Ub overexpression has an effect on ZIKV-mediated APP stabilization is also determined. Because the ubiquitination process affects APP processing, ZIKV may influence the proteasome pathway, and through which, eventually affect proteolytic processing of APP. Whether P2 increases APP RNA is also examined. APP mRNA's alternation during ZIKV infection is also tested. However, given the nature of the P2 peptide, it is unlikely that P2 could enhance the mRNA levels of APP.
The P2 peptide offers a simple and effective system for APP stability studies. We assess the mechanism of ZIKV-mediated enhancement (
Outcomes: 1) An ZIKV P2 peptide may be interacting with APP; 2) APP processing may be affected by P2 peptide; or 3) APP processing and P2 interactions are correlated. With several alternative approaches, the mechanisms of P2 peptide and ZIKV to enhance APP expression are inferred. We predict that both P2 peptide and ZIKV interact with APP, alter and slow down the APP proteolytic process, and eventually increase APP expression. Other mechanisms may also be present and are examined in a systemic manner. The potential usage of the P2 peptide as a therapeutic agent is inferred.
Alternatives: If results related to P2 peptide are not satisfying, we use a ZIKV virion to address the same issue. In addition, we address whether alternation of endogenous protease target affects the ZIKV mediated APP stabilization.
Hypothesis: APP inhibits ZIKV replication in a systematic manner.
Rationale and Significance: APP is inducible in several organs after ZIKV infection (
APP has restricted expression in other organs/tissues. In addition, we used a very few DKO mice and found some interesting pathogenic outcomes (
First, we titrate ZIKV PRVABC59 on the new DKO (app−/−; stat2−/−) strain. Basically, three DKO mice (three females) are infected with ZIKV (vaginal injection) with three dosages (104, 105, 106 pfu) are used for vaginal injection. Mouse weights (every day) and virus titers in the blood (every two days) are monitored through 8 days. The dosage(s) that cause weight loss, viremia, but not killing the mice in first 8 days are chosen for further experiments. Second, adult male mice are inoculated with ZIKV (PRVABC59, 104 pfu) through s.c. injections and female mice are inoculated with ZIKV (dosage determined by titrations) via vaginal inoculation as described (
Outcomes: A) The role of APP in ZIKV infection and pathogenesis is systematically revealed, and possibly as an inhibitor. Novel pathogenic phenotypes may be identified as in
Alternatives: A) The standard 6-8-week old mice are chosen for the experiments. If the results, especially the pathogenesis, are moderate, we choose older mice (4-6-month olds), because the DKO mice for initial test were all aged ones (>4-month-old, retired mice) (
Hypothesis: APP inhibits ZIKV replication in fetal brains, thereby decreasing the risk of microcephaly.
Rationale: APP is expressed in embryos. Fetal brains are readily infected by ZIKV. APP has a stronger anti-ZIKV effect in newborn mouse brains (P4 and P7) (
Significance: The role of APP in ZIKV-mediated fetal brain disruption is established, and a model for ZIKV-mediated brain damages, possible for microcephaly, is generated.
Fetal brains express APP and decent portion of NPCs/NSCs. It is possible that APP is also anti-ZIKV gene in fetuses and prevent the development of microcephaly.
To study the fetal brain changes during ZIKV infection of pregnant mice, we use the DKO (app−/−, stat2−/−) dams and heterozygous (app+/−, stat2−/−) sires that are obtained by crossing the DKO and stat2-null mice (
We: 1) measure the fetus body and head sizes. The fetal body sizes are measured by crown-rump length (CRL) and head sizes are obtained by occipito-frontal (OF) diameter; 2) determine the gene types of fetuses. Not only the fetuses, but also resorbed fetuses and conceptuses if present, are subject to genotyping; 3) examine fetal brain structural changes. Fetal brains are fixed by PFA. The gross morphology of the affected brains is visualized with the Nissl Stain. The thickness of marginal zone (MZ), cortical plate (CP), subplate (SP), intermediate zone (IZ), subventricular zone (SVZ), and ventricular zone (VZ) are measured; 4) determine viral load in the brains. A portion of the fetal brains is used for viral loads detections; 5) determine the percentage of cells infected by ZIKV, apoptosis and proliferation. The brain sections are stained with ZIKV, activated Caspase 3, phospho-Histone H3 antibodies, and DAPI simultaneously. The number of the positive cells is counted and quantified; 6) determine the nature of brain cells: NeuN (a marker for neurons), TBR1 (early and immature neurons marker), Nestin and Sox2 (Neural progenitor/stem cells marker) are used for staining. The positive cells are counted and thickness of the layers stained with markers are quantified; 7) determine the identity of ZIKV-infected cells. Antibodies for ZIKV and specific markers (determined in 6) are used to determine the identity of ZIKV-infected cells. Due to emission overlaps, maximum 3 antibodies plus DAPI can be used for Confocal imaging studies. State-of-the-art imaging technology is used in UNL Microcopy Core Facility.
Outcomes: 1) The advantage of the design (
Alternatives: A) Timing for infection and dissection of fetuses may need to be adjusted because the proposed schedules are based on literatures and our data. B) Number of dams may need be increased if statistical power is not reached. C) Use of IFN-receptor antibodies as alternative. Basically, incrossed and pregnant dams (WT and APP-null) are treated with IFNAR antibody every two days, starting on the Day-1, relative to ZIKV infection. IFNAR-blocking mAb and its control are available commercially. Fetuses and placentas are harvested for studies. The analyses are the same as described above. Because pregnant mice with the antibody still made enough IFNs to partially control the infection, the best approach would be the use of DKO mice. D) IFNs cause intrauterine growth restriction (IUGR). As the role of APP is examined in an STAT2-null background, IUGR might not be a problem.
Together these experiments shed light on a novel intrinsic immunity against ZIKV. With the completion of the proposed experiments, we may establish the role of APP in ZIKV infection and pathogenesis and a model for microcephaly in rodents, which would represent a vertical advance in both ZIKV and microcephaly research. Nevertheless, the research may provide important information on how to prevent and treat ZIKV-related diseases. Moreover, the research may benefit brain diseases studies in general.
P2 interacts with APP and block APP metabolic processing, and reduce amyloid formation (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a Divisional of U.S. patent application Ser. No. 17/369,681 filed on Jul. 7, 2021, which claims the benefit of U.S. Patent Application No. 63/049,039 filed on Jul. 7, 2020. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
| Number | Date | Country | |
|---|---|---|---|
| 63049039 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17369681 | Jul 2021 | US |
| Child | 18417206 | US |
