Patent Application
20190307749
The present invention relates in general to the field of mitochondria-division inhibitors, and more particularly, to novel water-soluble inhibitors with improved activity.
Not Applicable.
The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 10, 2019, is named TECH1180_SeqList.txt and is 6 bytes in size.
Without limiting the scope of the invention, its background is described in connection with diseases or conditions in which excessive fragmentation of mitochondria or mitochondrial dysfunction is found.
Alzheimer's disease (AD) is a progressive, age-dependent illness, characterized by the progressive decline of memory, cognitive function, and changes in behavior and personality [1-4]. Currently, over 46.8 million people worldwide, including 5.4 million Americans, live with AD-related dementia, and this number is estimated to increase to 131.5 million by 2050 [5]. AD-related dementia has huge economic consequences, with the total worldwide medical cost of dementia in 2015 estimated at $818 billion. By 2018, AD, including AD-related dementia, is expected to become a trillion-dollar disease [5]. With lifespan increasing in humans, AD is headed towards becoming the major health concern of elderly persons. Currently, there are no drugs or agents that can delay disease progression in elderly individuals and in patients with AD. Further, there are no definitive peripheral biomarkers that indicate disease in its early stages so that effective therapeutics can be initiated.
AD is largely associated with synaptic damage, mitochondrial structural and functional changes, inflammatory responses, hormonal imbalance, cell cycle changes, and neuronal loss [6-8]. In addition, there are 2 major pathological hallmarks: intracellular neurofibrillary tangles and extracellular amyloid-β (Aβ) deposits in the learning and memory regions of brain [3,9]. Genetic mutations in APP, PS1, and PS2 genes cause about 1-2% of the total number of AD cases. Several factors contribute to late-onset AD, including lifestyle, diet, environmental exposure, stroke, traumatic brain injury and multiple genetic variants in genetic loci, including sortilin-related receptor 1 gene clusterin, the complement component receptor 1, CD2AP, CD33, EPHA1, and MS4A4/MS4A6E genes and the ApoE4 genotype [7-8].
Despite many efforts toward treating AD, a need remains for compositions for use in preventing and/or treating the effects of AD at the molecular level.
In one embodiment, the present invention includes a method for preventing or treating a disease or condition with excessive fragmentation of mitochondria or mitochondrial dysfunction comprising, consisting essentially of, or consisting of: identifying a subject suspected of needing treatment for excessive fragmentation of mitochondria or mitochondrial dysfunction; and administering to the subject with an amount of a mitochondrial division inhibitor 1 sufficient to prevent or treat the excessive fragmentation of mitochondria or mitochondrial dysfunction. In one aspect, the mitochondrial division inhibitor 1 comprises a phenolic OH group that enhances antioxidant/anti-inflammatory and water solubility. In another aspect, the mitochondrial division inhibitor 1 is modified to be at least partially soluble in water. In another aspect, the disease or condition with excessive fragmentation of mitochondria or mitochondrial dysfunction is Alzheimer's, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, or Huntington's Disease. In another aspect, the method further comprises one or more pharmaceutically acceptable excipients, fillers, salts, or buffers. In another aspect, the disease is not associated with epilepsy and seizures, ischemia/reperfusion injury, oxygen glucose deprivation, or conditions associated with endosome aggregation and vesicle fusion during exocytosis. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject at a dose of 1 to 120 mg/day/person. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject at a dose of 10-60 mg daily. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject via oral or parenteral administration. In another aspect, the mitochondrial division inhibitor 1 is 3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone, 3-(2,4-Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone, or 3-(2,4-dichloro-5-hydroxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is a racemate, enantiomer, diastereomer, a mixture of enantiomer or a mixture of diastereomer. In another aspect, a pharmaceutically acceptable salt of mitochondrial division inhibitor 1 or active derivative thereof is formed from at least one of: an organic acid selected from formic acid, acetic acid, propionic acid, maleic acid, fumaric acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, malonic acid, oxalic acid, mandelic acid, glycolic acid, phtalic acid, benzenesulphonic acid, toluenesulphonic acid, naphtalenesulphonic acid, or, methanesulphonic acid. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is on the form of a tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, emulsion, or formulated for intravenous administration.
In another embodiment, the present invention includes a method of treating or preventing memory loss in a subject suffering from a memory loss-related disease or aging, comprising, consisting essentially of, or consisting of, administering an effective amount of an amount of a mitochondrial division inhibitor 1 or active derivative thereof sufficient to prevent or treat the excessive fragmentation of mitochondria or mitochondrial dysfunction. In one aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject at a dose of 1 to 120 mg/day/person. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject at a dose of 10-60 mg daily. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is administered to the subject via oral or parenteral administration. In another aspect, the mitochondrial division inhibitor 1 is 3-(2,4-Dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone, 3-(2,4-Dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone, or 3-(2,4-dichloro-5-hydroxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is a racemate, enantiomer, diastereomer, a mixture of enantiomer or a mixture of diastereomer. In another aspect, a pharmaceutically acceptable salt of mitochondrial division inhibitor 1 or active derivative thereof is formed from at least one of: an organic acid selected from formic acid, acetic acid, propionic acid, maleic acid, fumaric acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, malonic acid, oxalic acid, mandelic acid, glycolic acid, phtalic acid, benzenesulphonic acid, toluenesulphonic acid, naphtalenesulphonic acid, or, methanesulphonic acid. In another aspect, the memory loss-related disease is dementia. In another aspect, the dementia is Alzheimer's disease. In another aspect, a reduction in memory loss is in a cognitively normal older adult. In another aspect, the mitochondrial division inhibitor 1 or active derivative thereof is on the form of a tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, emulsion, or formulated for intravenous administration.
In another embodiment, the present invention includes a method for protecting a subject from excessive fragmentation of mitochondria or mitochondrial dysfunction in neural cells comprising, consisting essentially of, or consisting of: identifying a subject suspected of needing treatment for excessive fragmentation of mitochondria or mitochondrial dysfunction in neurons, wherein the subject is suspected of having or being at risk for Alzheimer's Disease; and administering to the subject with an amount of a mitochondrial division inhibitor 1 sufficient to prevent the excessive fragmentation of mitochondria or mitochondrial dysfunction to prevent Alzheimer's Disease.
In another embodiment, the present invention includes a partially water-soluble mitochondrial division inhibitor 1 having the formula 3-(2,4-dichloro-5-hydroxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
The present invention shows the protective and therapeutic effects of mitochondrial division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Mdivi1 is hypothesized to reduce excessive fragmentation of mitochondria and mitochondrial dysfunction in AD neurons. Very little is known about whether Mdivi1 can confer protective effects in AD. In these studies, the inventors sought to determine the protective effects of Mdivi1 against amyloid-β (Aβ)- and mitochondrial fission protein, dynamin-related protein 1 (Drp1)-induced excessive fragmentation of mitochondria in AD progression. The inventors also studied preventive (Mdivi1+Aβ42) and intervention (Aβ42+Mdivi1) affects against Aβ42 in N2a cells. Using real-time RT-PCR and immunoblotting analysis, the inventors measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis and synaptic genes. The inventors also assessed mitochondrial function by measuring H202, lipid peroxidation, cytochrome oxidase activity, and mitochondrial ATP. MTT assays were used to assess the cell viability. Aβ42 was found to impair mitochondrial dynamics, lower mitochondrial biogenesis, lower synaptic activity, and lower mitochondrial function. On the contrary, Mdivi1 enhanced mitochondrial fusion activity, lowered fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in Mdivi1-treated cells. Interestingly, Mdivi1 pre- and post-treated cells treated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability, mitochondrial dynamics, mitochondrial biogenesis, and synaptic activity. The protective effects of Mdivi1 were stronger in N2a+Aβ42 pre-treated with Mdivi1, than in N2a+Aβ42 cells than Mdivi1 post-treated cells, indicating that Mdivi1 works better in prevention than treatment in AD like neurons.
A dosage unit for use in a method of the present invention includes a mitochondrial division inhibitor 1 or active derivative thereof of the present invention, may be a single compound or mixtures thereof with other compounds, e.g., a potentiator. The compounds may be mixed together, form ionic or even covalent bonds. The mitochondrial division inhibitor 1 or active derivative thereof of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the mitochondrial division inhibitor 1 or active derivative thereof of the present invention to a patient in need of therapy. The mitochondrial division inhibitor 1 or active derivative thereof may also be administered as any one of known salt forms.
The mitochondrial division inhibitor 1 or active derivative thereof is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices. Depending on the best location for administration, the mitochondrial division inhibitor 1 or active derivative thereof may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the mitochondrial division inhibitor 1 or active derivative thereof may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and/or location of administration selected.
Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, N.Y., 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions incorporated herein by reference.
For example, the mitochondrial division inhibitor 1 or active derivative thereof may be included in a tablet. Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and/or melting agents. For example, oral administration may be in a dosage unit form of a tablet, gelcap, caplet or capsule, the active drug component being combined with a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like. Suitable binders for use with the present invention include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants for use with the invention may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like. Disintegrators may include: starch, methyl cellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.
The mitochondrial division inhibitor 1 or active derivative thereof may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles, whether charged or uncharged. Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like.
The mitochondrial division inhibitor 1 or active derivative thereof may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug. Such polymers may include: polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like. Furthermore, the mitochondrial division inhibitor 1 or active derivative thereof may be coupled one or more biodegradable polymers to achieve controlled release of the mitochondrial division inhibitor 1 or active derivative thereof, biodegradable polymers for use with the present invention include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.
In one embodiment, gelatin capsules (gelcaps) may include the mitochondrial division inhibitor 1 or active derivative thereof and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Like diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained-release formulations to provide for a range of release of medication over a period of minutes to hours. Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere. An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.
For oral administration in a liquid dosage form, the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.
Liquid dosage forms for oral administration may also include coloring and flavoring agents that increase patient acceptance and therefore compliance with a dosing regimen. In general, water, a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions. Solutions for parenteral administration include generally, a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts. Antioxidizing agents such as sodium bisulfite, sodium sulfite and/or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA may also be included to increase stability. In addition, parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl-paraben, and/or chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.
For direct delivery to the nasal passages, sinuses, mouth, throat, esophagous, trachea, lungs and alveoli, the mitochondrial division inhibitor 1 or active derivative thereof may also be delivered as an intranasal form via use of a suitable intranasal vehicle. For dermal and transdermal delivery, the mitochondrial division inhibitor 1 or active derivative thereof may be delivered using lotions, creams, oils, elixirs, serums, transdermal skin patches and the like, as are well known to those of ordinary skill in that art. Parenteral and intravenous forms may also include pharmaceutically acceptable salts and/or minerals and other materials to make them compatible with the type of injection or delivery system chosen, e.g., a buffered, isotonic solution. Examples of useful pharmaceutical dosage forms for administration of mitochondrial division inhibitor 1 or active derivative thereof may include the following forms.
Capsules. Capsules may be prepared by filling standard two-piece hard gelatin capsules each with 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.
Soft Gelatin Capsules. A mixture of active ingredient is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.
Tablets. A large number of tablets are prepared by conventional procedures so that the dosage unit was 100-500 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
To provide an effervescent tablet appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
Injectable solution. A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in deionized water and mixed with, e.g., up to 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized using, e.g., ultrafiltration.
Suspension. An aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.
For mini-tablets, the active ingredient is compressed into a hardness in the range 6 to 12 Kp. The hardness of the final tablets is influenced by the linear roller compaction strength used in preparing the granulates, which are influenced by the particle size of, e.g., the monosodium hydrogen carbonate and sodium hydrogen carbonate. For smaller particle sizes, a linear roller compaction strength of about 15 to 20 KN/cm may be used.
Kits. The present invention also includes pharmaceutical kits useful, for example, for the treatment of cancer, which comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of mitochondrial division inhibitor 1 or active derivative thereof. Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, may also be included in the kit. It should be understood that although the specified materials and conditions are important in practicing the invention, unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized.
Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
As used herein, the term “chewable” refers to semi-soft, palatable and stable chewable treat without addition of water. It should be appreciated to the skilled artisan that a chewable composition will be stable and palatable, fast disintegrating, semi-soft medicated chewable tablets (treats) by extrusion without the addition of extraneous water. A soft chewable tablets does not harden on storage and are resistant to microbial contamination. A semi-soft chewable contain a blend of any one or more of binders, flavours, palatability enhancers, humectants, disintegrating agents, non-aqueous solvents, and diluents that are plasticized with liquid plasticizers, such as glycols and polyols to make them ductile and extrudable. The chewable can be made by extrusion, e.g., including fats or lipids as plasticizers and binding agents, is manufactured in the absence of added water, uses plasticizers to replace water in extrudable matrices, contains humectants to maintain the extrudable chew in a pliant and soft state during its shelf life, or any combination thereof. The chewable form may be provided in conjunction with one or more flavorants and/or taste masking agents that improve the taste of the formulation greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90%. The chewable can include the active agent and the ion exchange resin to enhance taste masking.
For topical administration, the composition can be incorporated into creams, ointments, gels, transdermal patches and the like. The composition can also be incorporated into medical dressings, for example wound dressings e.g. woven (e.g. fabric) dressings or non-woven dressings (e.g. gels or dressings with a gel component). The use of alginate polymers in dressings is known, and such dressings, or indeed any dressings, may further incorporate the alginate oligomers of the invention.
Generally, the formula of the mitochondrial division inhibitor 1 is:
As taught herein, the present invention also includes an improvement to the solubility of the mitochondrial division inhibitor 1 (Mdivi1), which is an active derivative in which the modified Mdivi1 structure has a phenolic OH group that enhances antioxidant/anti-inflammatory properties and increases solubility. The at least partially water-soluble inhibitor has the formula: 3-(2,4-dichloro-5-hydroxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one. One advantage of the partially water-soluble Mdivi1 is that the use of the toxic compound dimethyl sulfoxide (DMSO) can be avoided. Because of safety concerns, clinical trials with DMSO have been stopped. DMSO is known to have toxicity of the eye, induces headaches, causes a burning and itching on contact with the skin, and triggers strong allergic reactions.
The present inventors recognized that mitochondrial abnormalities are largely involved in AD progression. For example, AD is associated with: (1) increased free radical production, lipid peroxidation, oxidative DNA damage, oxidative protein damage, and decreased ATP production in postmortem AD brains, compared to brains from age-matched healthy subjects [10-14]; in AD transgenic mice; and in cell lines that express mutant APP or cells treated with Aβ [15-16], (2) studies of mitochondrial enzyme activities found decreased levels of cytochrome oxidase activity, pyruvate dehydrogenase, and α-ketodehydrogenase in fibroblasts, lymphoblasts, and postmortem brains from AD patients, compared to neurons, fibroblasts, and lymphoblasts from age-matched healthy subjects [16], (3) mitochondrial DNA changes were higher in postmortem brain tissue from AD patients and aged-matched healthy subjects, compared to DNA changes in postmortem brain tissue from young, healthy subjects, suggesting that the accumulation of mitochondrial DNA in AD pathogenesis is age-related [17-18], (4) several groups investigated mitochondrial gene expressions in postmortem AD brains and in brain specimens from AD transgenic mice [19-21]. It was found that mitochondrially encoded genes abnormally expressed in the brains AD patients and AD mice. A recent, time-course global gene expression study in an AD mouse model (Tg2576) and age-matched non-transgenic littermates revealed an up-regulation of mitochondrial genes in the Tg2576 mice, suggesting that mitochondrial metabolism is impaired by mutant APP/Aβ and that the up-regulation of mitochondrial genes may be a compensatory response to mutant APP/Aβ [20]; further, Manczak and colleagues found an abnormal expression of mitochondrially encoded genes in postmortem AD brains compared to the brains of healthy subjects, suggesting impaired mitochondrial metabolism in AD [22], 5) recent mitochondrial studies in brain tissue from AD patients and neuronal cells expressing mutant APP found that Aβ fragments mitochondria and causes structural changes in neurons from AD patients [21,23-27].
Recent Aβ and mitochondrial studies found impaired mitochondrial dynamics (excessive mitochondrial fragmentation and reduced fusion) in AD postmortem brains and AD cell and mouse models [21,23-25,26-31]. Studies by the inventors revealed that Aβ interacts with Drp1, with a subsequent increase in free radical production, which in turn activates Drp1 and Fis1, and causes excessive mitochondrial fragmentation, defective transport of mitochondria to synapses, low synaptic ATP, and synaptic dysfunction in AD neurons [21,24]. Given the increase in free radical production and excessive fragmentation in mitochondria that are involved in AD, the inventors hypothesize that drugs capable of reducing free radicals or decreasing excessive mitochondrial fragmentation may be effective therapeutic approaches to treat AD.
Excessive mitochondrial fragmentation is well documented in neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's, leading to impaired mitochondrial dynamics, defective axonal transport, low synaptic ATP and, ultimately, synaptic damage [32,33]. Based on excessive mitochondrial fragmentation, impaired mitochondrial dynamics, defective axonal transport, low synaptic ATP, and synaptic damage that are present in AD, it has been proposed that mitochondrial division inhibitors are potential candidates to treat excessive mitochondrial fragmentation and its associated factors in AD progression and pathogenesis.
To identify mitochondrial fission inhibitors, several groups have independently screened chemical libraries and have found several inhibitors: Mdivi1 [34], Dynasore [35], and P110 [36]. Among these, Mdivi1 has been extensively investigated with experimental rodent models of epilepsy and seizures [37], ischemia/reperfusion injury [38-41], oxygen glucose deprivation [42], and such conditions as aggregation of endosomes and vesicle fusion during exocytosis [43]. In all of these diseased states and conditions, Mdivi1 was found to have beneficial effects on affected tissues and cells, by reducing excessive mitochondrial fission and maintaining the fission-fusion balance in mitochondria and the normal functioning of cells.
Mdivi1 is a cell-permeable, selective mitochondrial fission inhibitor; its molecular weight is 353.22. It inhibits GTPase Drp1 activity by blocking the self-assembly of Drp1, resulting in reversible formation of elongated and tubular mitochondria in wild-type cells [34]. However, it is unclear whether Mdivi1 reduces Aβ-induced excessive mitochondrial fragmentation, maintains and/or enhances mitochondrial function and synaptic activities in Aβ-treated N2a cells.
In the present study, the inventors determined the protective effects of Mdivi1 against Aβ-induced excessive mitochondrial fragmentation and synaptic toxicities in mouse neuroblastoma (N2a) cells. Using Aβ42 peptide and Mdivi1 and mouse neuroblastoma (N2a) cells, the present inventors measured (1) mRNA and protein levels of mitochondrial fission and fusion genes, biogenesis genes, and synaptic genes; (2) mitochondrial function by measuring H2O2, lipid peroxidation, cytochrome oxidase activity, and mitochondrial ATP; and (3) cell viability.
Chemicals and Reagents: The Aβ42 peptide was purchased from Anaspec (Fremont, Calif., USA); Water soluble Mdivi1 was made in our laboratory and N2a cells were purchased from American Type Culture Collection (ATCC) (Manassas, Va., USA). Dulbecco's Modified Eagle Medium (DMEM) and Minimum Essential Medium (MEM), penicillin/streptomycin, Trypsin-EDTA, and fetal bovine serum were purchased from GIBCO (Gaithersberg, Md., USA).
Tissue culture: The N2a cells were grown for 3 days in a serum-free medium (1:1 mixture of DMEM and OptiMEM, plus penicillin and streptomycin [Invitrogen, Carlsbad, Calif., USA]) until the cells developed neuronal processes. As shown in
The N2a cells from all 5 groups (4 treatment groups and 1 control group) were harvested, and their cell pellets were collected and used for quantitative real-time RT-PCR, immunoblotting analysis of mitochondrial and synaptic proteins, and mitochondrial functional assays for hydrogen peroxide production, lipid peroxidation, cytochrome c oxidase activity, and MTT determination. Isolated mitochondria from N2a cells from all 5 groups were used to determine mitochondrial ATP as described in Reddy et al. [6]. As shown in
Quantitative real-time RT-PCR: Using the reagent TriZol (Invitrogen), total RNA was isolated from cell pellets from the 5 N2a cell groups (
The mRNA transcript level was normalized against β-actin and the GAPDH at each dilution. The standard curve was the normalized mRNA transcript level, plotted against the log-value of the input cDNA concentration at each dilution. To compare β-actin, GAPDH, and relative quantification was performed according to the (CT) method (Applied Biosystems). Briefly, this method involved averaging triplicate samples, which were taken as the CT values for β-actin, GAPDH, and neuroprotective markers. β-actin normalization was used in the present study because the β-actin CT values were similar for the control and the 4 treatment groups in terms of mitochondrial dynamics, biogenesis, and synaptic genes. The ΔCT-value was obtained by subtracting the average β-actin CT value from the average CT-value of the synaptic mitochondrial ETC genes and the mitochondrial structural genes. The ΔCT of untreated cells was used as the calibrator. The fold change was calculated according to the formula 2{circumflex over ( )}-(Δ ΔCT), where ΔΔCT is the difference between ΔCT and the ΔCT calibrator value. To determine the statistical significance of mRNA expression, the CT value differences between the untreated cells and other experimental groups of cells were used in relation to β-actin normalization. Statistical significance was calculated, using one-way ANOVA. Fold changes of mRNA were compared 2 ways—comparison 1, untreated N2a cells with 1) N2a+Mdivi1, 2) N2a+Aβ42, 3) N2a+Aβ42+Mdivi1, 4) N2a+Mdivi1+Aβ42, and comparison 2, N2a+Aβ42 with 1) N2a+Aβ42+Mdivi1 and 2) N2a+Mdivi1+Aβ42.
Immunoblotting analysis: To determine the effects of Mdivi1 and Aβ42 at the protein levels of mitochondrial dynamics and biogenesis and synaptic genes that exhibited altered mRNA expression, the inventors performed immunoblotting analyses of protein lysates isolated from cell pellets of all 5 groups of cells (
Measurement of soluble Aβ42 levels in untreated N2a cells and N2a cells treated with Mdivi1. The cell pellets were washed several times with PBS buffer and washed pellets were used and measured soluble Aβ42 levels in the N2a cells+Aβ42, Aβ42+Mdivi1, and Mdivi1+Aβ42 treatment groups following method described in Manczak et al [23]. Briefly, protein lysates from untreated control N2a cells and lysates from 4 treatment groups were homogenized in a Tris-buffered saline (pH 8.0) containing protease inhibitors (20 mg/ml pepstatin A, aprotinin, phophsoramidon, and leupeptin; 0.5 mM phenylmethanesulfonyl fluoride and 1 mM ethyleneglycol-bis(flaminoethyl ether)-NN tetraacetic acid). Samples were sonicated briefly and centrifuged at 10,000 g for 20 min at 4° C. The soluble fraction was used to determine the soluble Aβ by ELISA. For each sample, Aβ1-40 and Aβ1-42 were measured with commercial colorimetric ELISA kits (Biosource International, Camarillo, Calif., USA) that were specific for each species. A 96-well plate reader was used, following the manufacturer's instructions. Each sample was run in duplicate. Protein concentrations of the homogenates were determined following the BSA method, and Aβ was expressed as pg Aβ/mg protein.
Mitochondrial functional assays. H2O2 production: Using an Amplex® Red H2O2 assay kit (Molecular Probes, Eugene, Oreg., USA), H2O2 production was measured using cell pellets, as described in Manczak and Reddy [44]. Briefly, H2O2 production was measured in the protein lysates prepared from cell pellets of control untreated N2a cells and 4 treatment groups (
Lipid peroxidation assay: Lipid peroxidates are unstable indicators of oxidative stress in the brain. The final product of lipid peroxidation is 4-hydroxy-2-nonenol (HNE), which was measured in the N2a cell lysates prepared from cell pellets of control and treatment groups. The inventors used an HNE-His ELISA kit (Cell BioLabs, Inc., San Diego, Calif., USA), as described in Manczak and Reddy [44]. Briefly, freshly prepared protein as added to a 96-well protein binding plate and incubated overnight at 4° C. The protein then washed 3 times with a buffer. After the last wash, the anti-HNE-His antibody was added to the protein in the wells, incubated for 2 hours at room temperature, and then washed again 3 times. The samples were then incubated with a secondary antibody conjugated with peroxidase for 2 hours at room temperature, followed by incubation with an enzyme substrate. Optical density was measured (at 450 nm) to quantify the level of HNE. Lipid peroxidation levels were compared 2 ways—comparison 1, untreated N2a cells with 1) N2a+Mdivi1, 2) N2a+Aβ42, 3) N2a+Aβ42+Mdivi1, 4) N2a+Mdivi1+Aβ42, and comparison 2, N2a+Aβ with 1) N2a+Aβ42+Mdivi1 and 2) N2a+Mdivi1+Aβ42.
Cytochrome c oxidase activity: Cytochrome oxidase activity was measured in each of the 5 groups of N2a cells. Enzyme activity was assayed spectrophotometrically using a Sigma Kit (Sigma-Aldrich) following manufacturer's instructions. Briefly, 2 μg protein lysate was added to 1.1 ml of a reaction solution containing 50 μl 0.22 mM ferricytochrome c that was fully reduced by sodium hydrosulphide, Tris-HCl (pH 7.0), and 120 mM potassium chloride. The decrease in absorbance at 550 mM was recorded for 1-min reactions at 10-sec intervals. Cytochrome oxidase activity was measured according to the following formula: mU/mg total mitochondrial protein=(A/min sample−(A/min blank)×1.1 mg protein×21.84). The protein concentrations were determined following the BCA method. Cytochrome oxidase activity levels were compared 2 ways—comparison 1, untreated N2a cells with 1) N2a+Mdivi1, 2) N2a+Aβ42, 3) N2a+Aβ42+Mdivi1, 4) N2a+Mdivi1+Aβ42, and comparison 2, N2a+Aβ42 with 1) N2a+Aβ42+Mdivi1 and 2) N2a+Mdivi1+Aβ42.
ATP levels: ATP levels were measured in N2a cell mitochondria from the treatment groups using an ATP determination kit (Molecular Probes). A bioluminescence assay was used, based on the reaction of ATP with recombinant firefly luciferase and its substract luciferin. Luciferase catalyzes the formation of light from ATP and luciferin. It is the emitted light that is linearly related to the concentration of ATP, which is measured with a luminometer. ATP levels were measured from mitochondrial pellets using a standard curve method. ATP levels were compared 2 ways—comparison 1, untreated N2a cells with 1) N2a+Mdivi1, 2) N2a+Aβ42, 3) N2a+Aβ42+Mdivi1, 4) N2a+Mdivi1+Aβ42, and comparison 2, N2a+Aβ42 with 1) N2a+Aβ42+Mdivi1 and 2) N2a+Mdivi1+Aβ42.
Statistical considerations. Statistical analyses were conducted for mitochondrial structural and functional parameters in the N2a cells from the 5 experimental groups, using one-way ANOVA with Dunnett correction. The parameters included H2O2, cytochrome oxidase activity, lipid peroxidation, ATP production, and cell viability. To determine the effect of Mdivi1 on N2a cells, in the absence and presence of Aβ42, the inventors analyzed and compared data in 2 ways—comparison 1, untreated N2a cells with 1) N2a+Mdivi1, 2) N2a+Aβ42, 3) N2a+Aβ42+Mdivi1, 4) N2a+Mdivi1+Aβ42, and comparison 2, N2a+Aβ with 1) N2a+Aβ+Mdivi1 (curative) and 2) N2a+Mdivi1+Aβ42 (preventive).
mRNA expressions of mitochondrial dynamics genes. Amyloid-β42 treatment: In the N2a cells treated with Aβ42 compared to untreated N2a cells, mRNA expression levels were significantly higher: in the fission Drp1 by 1.4 fold (P=0.02) and Fis1 by 1.4 fold (P=0.03) (Table 3). In contrast, mRNA expression levels of mitochondrial fusion genes were lower but not significant—Mfn1 by −1.2 fold, Mfn2 by −1.3 fold, and Opa1 by −1.2 fold. These findings indicate the presence of abnormal mitochondrial dynamics in cells treated with Aβ.
Mdivi1: The mRNA levels of N2a cells treated with Mdiv1 were significantly lower in the fission genes Drp1 (1.5-fold decrease, P=0.01 and Fis1 (1.3-fold decrease) and higher for the fusion genes Mfn1 by 1.3 fold, Mfn2 by 1.2 fold, and Opa1 by 1.2 fold (Table 3).
Treatment with Aβ42 and Mdivi1: In the N2a cells treated with Aβ42 and then treated with Mdivi1, the mRNA levels were unchanged for Drp1 and Fis1 and for Mfn1, Mfn2 and Opa1 and CypD, compared to the mRNA levels of untreated N2a cells (Table 3). The mRNA levels of N2a cells treated with Mdivi1 and then treated with Aβ42 did were significantly higher for the fusion genes Mfn1 by 2.1 fold (P=0.01), Mfn2 by 1.7 fold (P=0.03), and Opa1 by 1.9 fold (P=0.01) (Table 3).
Mitochondrial biogenesis genes. Aβ42: To determine the effects of Aβ42 and Mdivi1 on mitochondrial biogenesis genes, mRNA expression levels of PGC1a, Nrf1, Nrf2, and TFAM genes were measured. Significantly lower mRNA expressions were found in the biogenesis genes from N2a cells treated with Aβ42 relative to the mRNA expression level of untreated cells:—PGC1α by 5.8 fold (P=0.001), Nrf1 by 2.0 fold (P=0.01), Nrf2 by 2.1 fold (P=0.01), and TFAM by 2.5 fold (P=0.01) (Table 3).
Mdivi1: mRNA levels were significantly increased for PGC1α by a 2.2-fold (P=0.01), Nrf1 by a 2.2 fold (P=0.01), Nrf2 by 1.6 fold (P=0.03), and TFAM by a 1.5 fold (P=0.03) in Mdivi-treated cells relative to untreated cells (Table 3). These observations indicate that Mdivi1 increases mitochondrial biogenesis activity.
Aβ42+Mdivi1: In cells treated with Aβ42 first and then treated with Mdivi1, mRNA expression levels were unchanged for Nrf1, Nrf2 and only slightly higher for PGC1α (by 1.4 fold) and TFAM (1.2 fold) (Table 3).
Mdivi1+Aβ42: In cells treated with Mdivi1 and then treated with Aβ42, levels of mRNA expression were slightly higher for biogenesis genes: PGC1α by 1.1 fold, Nrf1 by 1.3 fold, Nrf2 by 1.3, and TFAM by 1.3 (Table 3). These results suggest that Mdivi1 treatment prevented Aβ42-induced biogenesis toxicity.
Synaptic genes. Aβ42: In cells treated with Aβ42 compared to untreated cells, mRNA expression levels were lower for synaptophysin by 1.4 fold (P=0.04) and PSD95 by 2.6 fold (P=0.004), indicating that Aβ42 reduces synaptic activity (Table 3).
Mdivi1: mRNA levels were significantly higher for PSD95 5.1 fold (P=0.004) and higher for synaptophysin by 1.3 fold in the Mdivi1-treated cells (Table 3). These findings indicate that Mdivi1 boosts synaptic activity in healthy cells.
Aβ42+Mdivi1: In cells treated with Aβ42 and then treated with Mdivi1, mRNA levels were significantly higher for PSD95 by 4.8 fold (P=0.001) and slightly higher for synaptophysin 1.2 fold (Table 3). These observations suggest that Mdivi1 rescued synaptic activity from Aβ42-induced toxicity.
Mdivi1+Aβ: In cells treated with Mdivi1 and then treated with Aβ42, significantly higher mRNA expression levels were found for synaptophysin by 1.7 fold (P=0.01) and PSD95 by 1.5 fold (P=0.04) (Table 3), indicating that Mdivi1 prevented Aβ42-induced synaptic activity.
Comparison to Aβ42-treated N2a cells: As shown in Table 3, mRNA expression levels of fission genes were lower in N2a cells treated with Aβ42+Mdivi1 (Drp1 by 1.5 fold, P=0.03; Fis1 by 1.7 fold, P=0.02), and with Mdivi1+Aβ42 (Drp1 by 1.5 fold, P=0.02; Fis1 by 1.6 fold, P=0.03) relative to the expression levels of fission genes in N2a cells treated only with Aβ42. In contrast, fusion genes were higher in the N2a cells treated with Aβ42+Mdivi1 (Mfn1 by 1.6 fold, P=0.04; Mfn2 by 1.6 fold, P=0.03; Opa1 by 1.3) and Mdivi1+Aβ42 (Mfn1 by 2.6 fold, P=0.003; Mfn2 by 2.2 fold, P=0.01; Opa1 by 2.3 fold, P=0.002) than the N2a cells treated only with Aβ42.
Mitochondrial biogenesis genes were greater in the N2a cells treated with Aβ42+Mdivi1 (PGC1α by 8.1 fold, P=0.0001; Nrf1 by 2.0 fold, P=0.01, Nrf2 by 2.0 fold, P=0.01, TFAM by 2.9 fold, P=0.004) and Mdivi1+Aβ42 (PGC1α by 6.5 fold, P=0.001; Nrf1 by 2.7 fold, P=0.003; Nrf2 by 2.7 fold, P=0.004; TFAM by 3.2 fold, P=0.002) than in the N2a cells treated only with Aβ42, indicating that Mdivi1 increases Aβ42-induced reduced biogenesis activity. Similarly, synaptic genes were greater in N2a cells treated with Aβ42+Mdivi1 (synaptophysin by 1.5 fold, P=0.04 and PSD95 by 8.6 fold, P=0.0002) and Mdivi1+Aβ42 (synaptophysin 2.2 fold, P=0.01 and PSD95 by 3.8 fold, P=0.003) than in N2a cells treated only with Aβ42 (Table 3).
Immunoblotting analysis. To determine the effects of Aβ42 and Mdiv1 on mitochondrial proteins, the inventors quantified mitochondrial proteins in 3 independent treatments of N2a cells with Aβ42, Mdivi1, Aβ42+Mdivi1, and Mdivi1+Aβ42.
Comparison to untreated N2a cells: In N2a cells treated with Aβ42, significantly higher increased proteins levels were found for Drp1 (P=0.04) and Fis1 (P=0.01) (
Drp1 (P=0.001) and Fis1 (P=0.003) protein levels were significantly lower, in contrast to Mfn1 (P=0.01), Mfn2 (P=0.01) and OPA1 (P=0.01) protein levels were significantly higher in N2a cells treated with Mdivi1 (
Unchanged protein level for Drp1 and Fis1 were in N2a cells treated with Aβ42+Mdivi1, compared to levels of these proteins in untreated N2a cells (
Comparison to Aβ42-treated cells: As shown in
Also significantly higher levels of PGC1α (P=0.002), Nrf1 (P=0.02) and TFAM (P=0.02) were found in N2a cells treated with Aβ42+Mdivi1 relative to Aβ42 treated N2a cells, similar to PGC1α (P=0.001), Nrf1 (P=0.04), Nrf2 (P=0.001), and TFAM (P=0.01) N2a cells that were treated with Mdivi1+Aβ42 (
Mdivi1 and levels of Aβ42. To determine whether Mdivi1 lowers Aβ42 levels in N2a cells, using Sandwich ELISA the inventors measured Aβ42 levels in N2a cells treated with Aβ42+Mdivi1 (curative) and Mdivi1+Aβ42 (preventive). As shown in
These observations indicate that Mdivi1 reduces Aβ42 levels via mitochondrial dynamics pathway since Aβ42 was found to be lower in Mdivi1+Aβ42 and Aβ42+Mdivi1 proteins, regardless of the sequence of treatment, as long as Mdivi1 was involved in the treatment. Also, since Aβ42 were found to be higher in N2a cells, regardless of the sequence of treatment, as long as Mdivi1 was involved in the treatment, mitochondrial fragmentation lowered, as did the level of free radicals.
Mitochondrial function. Parameters of mitochondrial function were studied in Aβ-treated N2a cells (n=4) to determine whether affects mitochondrial function and whether Mdivi1 confers preventive effects on Mdivi1 in the presence or absence of the Aβ42 peptide. The parameters included H2O2 production, cytochrome oxidase activity, lipid peroxidation, ATP production, and cell viability. The inventors compared the data 2 ways—in comparison 1—untreated N2a cells with Mdivi1, Aβ42, Mdivi1+Aβ42 and Mdivi1+Aβ42 (preventive) and in comparison 2—Aβ42 with Aβ42+Mdivi1 and Mdivi1+Aβ42 (curative).
H2O2 production: In comparison 1—as shown in
Cytochrome c oxidase activity: Significantly lower levels of cytochrome oxidase activity were found in N2a cells treated with Aβ42 (P=0.01) (
ATP production: Significantly lower levels of ATP were found in N2a cells that were treated with Aβ42 (P=0.01) (
Cell viability: Significantly lower levels of cell viability were found in N2a cells treated with Aβ42 (0.01) (
N2a cells treated with Aβ42 were found to impair mitochondrial dynamics (increased fission and decreased fusion), reduced biogenesis, and decreased synaptic activity and mitochondrial function. On the other hand, Mdivi1 reduced fission machinery and enhanced fusion activity and also increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in Mdivi1 treated cells. Interestingly, N2a cells treated with Mdivi1 and Aβ42 (Mdiv1+Aβ42 and Aβ42+Mdiv1) showed reduced mitochondrial dysfunction and maintained cell viability, mitochondrial dynamics, mitochondrial biogenesis, and synaptic activity
The protective effects of water soluble Mdivi1 were stronger in pre-treated cells than post-treated cells—in other words, Mdivi1 works better in prevention than treatment in AD like neurons, however, it is still able to treat AD. In the current study, impaired mitochondrial dynamics was evident in the Aβ42-treated N2a cells (Table 3). These findings agree with earlier studies [23,35-26,45] in which increased mitochondrial fission and reduced fusion were reported. Mitochondrial biogenesis genes were reduced in Aβ42-treated cells, indicating that Aβ42 reduces biogenesis activity. Synaptic activity was significantly reduced in Aβ42 treated N2a cells. Protein data confirmed mRNA changes, suggesting that Aβ42 affects both mRNA and proteins of mitochondria and synapses in AD like neurons. On the other hand, in cells treated with Mdivi1—mitochondrial biogenesis and synaptic genes were increased and mitochondrial fission genes were reduced and fusion genes were increased, indicating that Mdivi1 treatment is beneficial to cells. Further, when the inventors compared mRNA and protein data of N2a cells+Aβ42 with ‘Aβ42+Mdivi1 (curative)’ and ‘Mdivi1+Aβ42 (prevention)’, protective effects of preventive were stronger than Mdivi1—In other words, in the presence of Aβ42 in N2a cells, treatment of Mdivi1 before Aβ42 treatment did stronger than after Aβ42 followed by Mdivi1 treatment (see Table 3). These observations strongly suggest that Mdivi1 prevents mitochondrial structural, biogenesis and synaptic genes from expressing abnormally. Overall, Mdivi1 protects mitochondrial structure and mitochondrial function by regulating mitochondrial fission and fusion genes in AD.
Aβ42 levels were lower in N2a cells treated with Mdivi1. Interestingly, Mdivi1 reduces Aβ42 in both pre- and post-Mdivi1 treated cells in the presence of Aβ42. Based on current study findings, it appears that Mdivi1 may reduce excessive mitochondrial fragmentation and increase mitochondrial fusion activity, leading to reduced production of mitochondria-generated excessive free radicals, increased cell viability, and maintained mitochondrial/neuronal function. The toxicity caused by Aβ42 [16,20.46] is expected to be reduced by Mdivi1 treatment in N2a cells. However, further research is still needed to understand how Mdivi1 reduces Aβ42 levels in N2a cells.
In the current study, the inventors found mitochondrial function and cell viability were reduced in Aβ42 treated cells. These observations concur with previous studies on Aβ-induced defective mitochondrial function and cell viability [6,23,26,45-49]. On the other hand, Mdivi1-treated cells exhibited enhanced mitochondrial function—increased mitochondrial ATP, cytochrome oxidase activity and cell viability, and reduced free radicals and oxidative stress. These observations strongly suggest that Mdivi1 reduces cellular toxicity and boosts mitochondrial function.
Next, the inventors determined the protective effects of Mdivi1, particularly ‘reduced fragmentation of mitochondria’ and increased and/or maintained mitochondrial function and cell viability in the presence of Aβ42 in cells. The inventors measured mitochondrial functional parameters and cell viability in N2a cells treated with Mdivi1 in the presence or absence of Aβ42. As described in the results section, Aβ42-induced excessive mitochondrial fragmentation and defective mitochondrial function and cell viability were reversed in Mdivi1-treated cells. The reversal effect was stronger in Mdivi1 pre-treated cells than Mdivi1 post-treated cells (see Table 3), indicating that Mdivi1 acts as strong preventive drug for AD. These findings are consistent with mitochondrial and synaptic gene-expression and protein data. In the presence of Aβ42, Mdivi1 reduced free radicals and lipid peroxidation, and increased mitochondrial ATP and cytochrome oxidase activity and cell viability. Thus, these data clearly show that Mdivi1 protects cells from Aβ42 toxicity.
In summary, Mdivi1 treatment confers protective effects against Aβ42-induced mitochondrial and synaptic toxicities in N2a cells. Findings from our study may have implications for other neurological diseases, such as Huntington's [50-53], Parkinson's [54-56], multiple sclerosis [57], and ALS [58], in which excessive mitochondrial fragmentation is present.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.
For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
This application claims priority to U.S. Provisional Application Ser. No. 62/655,677, filed Apr. 10, 2018, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62655677 | Apr 2018 | US |
