Patent Application
20170209443
This invention relates to methods and pharmaceutical compositions for inhibiting or protecting against neuroinflammation using a Fyn tyrosine kinase inhibitor. In particular, it relates to use of the small molecule Fyn inhibitor saracatinib in the treatment of diseases, disorders, and conditions where neuroinflammation is implicated. The invention also relates to methods of using Fyn tyrosine kinase inhibitors and pharmaceutical compositions of this invention to treat neurodegenerative diseases, disorders, and conditions, such as Parkinson's disease.
Parkinson's disease (PD) is a neurodegenerative disorder affecting more than 1% of the population over the age of 60 in the US (Allam et al., 2005; West et al., 2005). PD is characterized clinically by severe motor symptoms including uncontrollable resting tremor, muscular rigidity, impaired postural reflexes, and bradykinesia, which vary between patients. These abnormalities can be accompanied by other symptoms, such as autonomic dysfunction, depression, and a general slowing of intellectual processes. Pathologically, PD is characterized by the marked degeneration of dopaminergic neurons in the substantia nigra pars compacta, which leads to the depletion of dopamine (DA) in its striatal projections, and of other brainstem neurons, with consequent disruption of the cerebral neuronal systems responsible for motor functions. This neurodegeneration is accompanied by the presence of cytoplasmic (Lewy bodies, LBs) and neuritic (Lewy neurites, LNs) inclusions in the surviving dopaminergic neurons and other affected regions of the central nervous system (CNS), but the mechanism underlying their formation is unclear, as is their pathogenic relevance.
PD is an essentially sporadic neurodegenerative disease whose pathogenesis remains largely unknown, despite years of intense research in an attempt to explain the complexity and the relative selectivity of dopaminergic neurodegeneration.
There are nine members of Src family of intracellular non-receptor tyrosine kinases. Five of them (Src, Fyn, Lck, Lyn, and Yes) are expressed in the central nervous system, but Src and Fyn are most highly expressed in the brain. Fyn activity, like that of other Src family kinases, is regulated by intramolecular interactions that depend on an equilibrium between tyrosine phosphorylation and dephosphorylation (Thomas et al., 1997, Ann. Rev. Cell & Dev. Biol. 13:513-609). In the basal state, catalytic activity is constrained by intramolecular interactions, such as engagement of the SH2 domain by a phosphorylated C-terminal Tyr 527. Disruption of these interactions by phosphorylation at Tyr 416 in the activation loop of the kinase domain and/or by dephosphorylation of Tyr 527 results in Fyn activation (Hunter, 1987, Cell, 49: 1-4).
Fyn has been localized to the post-synaptic density (PSD) fraction of the brain and amongst its substrates are receptors for the major excitatory transmitter glutamate. Fyn regulates glutamate receptor trafficking and synaptic plasticity (Nakazawa et al., 2001, J Biol Chem 276:693-699; Kojima et al., 1998, Learning & Memory (Cold Spring Harbor N.Y.) 5:429-445; Grant et al., 1992, Science 258:1903-1910; Prybylowski et al., 2005, Neuron 47:845-857). Specifically, Fyn phosphorylates the NMDA-type glutamate receptor subunits, NR2A and NR2B (Suzuki et al., 1995, Biochem Biophys Res Commun 216:582-588).
Inhibitors of a kinase of the Src family, such as dasatinib, bosutinib, saracatinib or ponatinib, are used in the treatment of cancer, in particular lung cancer. Approximately 85% of all lung cancer incidences are non-small cell lung cancer (NSCLC) (Jemal, Siegel et al., 2008). Non-small cell lung cancer (NSCLC) is one of the two main types of lung carcinoma, non-small cell (80.4%) and small-cell (16.8%) lung carcinoma, the classification being based on histological criteria. The non-small cell lung carcinomas have a similar prognosis and similar management and comprise three sub-types: squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma. Squamous cell lung carcinoma (31.1% of lung cancers) often starts near a central bronchus and commonly shows cavitation and necrosis within the center of the cancer. Adenocarcinoma (29.4% of lung cancers) mostly originates in peripheral lung tissue and is usually associated with smoking. Large cell lung carcinoma (10.7% of lung cancers) is a fast-growing form that develops near the surface of the lung. Common treatments of NSCLC include surgery, chemotherapy, and radiation therapy. In particular, NSCLC is treated with adjuvant chemotherapy (i.e. chemotherapy after surgery). Wu (2009) describes phosphorylation patterns in lung cancer (see Wu (2009) PloS ONE 4 (11) e7994).
It is an object of the current invention to provide novel pharmaceutical compositions and treatment protocols for Parkinson's disease and associated neuroinflammation through inhibition of Src intracellular non-receptor tyrosine kinases. Preferably treatment of Parkinson's disease without dementia, and more preferably treatment of Parkinson's without concomitant Amyloid-β accumulation.
The present invention provides methods and a pharmaceutical compositions comprising an effective, orally active amount of the Fyn tyrosine kinase inhibitor, for use in treating of Parkinson's and other related neurodegenerative disease.
The present invention further provides a method of treating or preventing inflammation in Parkinson's disease or other neurological disorder in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a Fyn inhibitor. The present invention further provides a method of preventing further neurodegeneration in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a Fyn inhibitor.
In certain embodiments, the Fyn inhibitor is selected from the group consisting of a nucleic acid, siRNA, antisense nucleic acid, ribozyme, peptide, antibody, small molecule, antagonist, aptamer, peptidomimetic, and any combinations thereof. In preferred embodiments, the Fyn small molecule inhibitor is selected from the group consisting of saracatinib, bosutinib, dasatinib, ponatinib, PP2, a salt, prodrug or solvate thereof, a derivative thereof, and any combinations thereof.
In certain embodiments, the composition is administered to the mammal by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes. In a preferred embodiment administration is oral.
In certain embodiments, the method further comprises administering to the mammal at least one additional agent that treats or prevents the Parkinson's or other neurological disease states, or other Fyn tyrosine kinase modulated diseases or disorders in the mammal. In yet other embodiments, the method further comprises administering to the mammal at least one additional agent that improves or prevents further neurodegeneration in the mammal. In yet other embodiments, the composition and at least one additional agent are co-formulated.
The present invention further provides a kit for preventing or treating an inflammation associated with Parkinson's disease or other neurological in a mammal, wherein the kit comprises a pharmaceutical composition of the invention, an applicator, and an instructional material for use thereof. In certain embodiments, the instructional material recites the amount of, and frequency with which, the composition is to be administered to the mammal.
In the preceding aspects of the invention, inhibitors of Fyn tyrosine kinase can be included in pharmaceutical compositions and administered to a mammal suffering from or at risk of a disease, disorder, or condition associated with Fyn tyrosine kinase activation, including but not limited to Parkinson's disease, Alzheimer's disease, Tourette's syndrome, schizophrenia, Huntington's disease, symptoms of attention deficit hyperactivity disorder, drug abuse and clinical depression. In a still further aspect, the invention concerns a method for treating neurodegeneration of neurons in a mammal in need thereof comprising administering to a mammal in need thereof a Fyn tyrosine kinase inhibitor, thereby providing neuroprotection. In another aspect, the pharmaceutical composition may be used in the treatment of diseases, disorders, or conditions associated with neurodegeneration. In one aspect, the neuron is a sympathetic, parasympathetic, or enteric, e.g. dorsal root ganglia neurons, motor neurons, and central neurons, e.g. neurons from the spinal cord.
As used herein, the term “Fyn tyrosine kinase inhibitor” includes any compound capable of downregulating, decreasing, reducing, suppressing or inactivating the amount and/or activity of Fyn tyrosine kinase. Generally, said inhibitors may be proteins, oligo- and polypeptides, polynucleotides, genes, lipid, polysaccharide, drugs, small chemical molecules, or other chemical moieties. Inhibitors for use with the invention may function to inhibit Fyn tyrosine kinase by any number of ways, including decreasing Fyn tyrosine kinase mRNA or protein levels or by blocking the activation of Fyn tyrosine kinase or its activity, for example, through inhibiting or decreasing proteolytic cleavage of Fyn tyrosine kinase using a Fyn tyrosine kinase peptide cleavage inhibitor. Compounds that decrease activity of Fyn tyrosine kinase downstream of Fyn tyrosine kinase in its pathway and decrease products or activity of Fyn tyrosine kinase targets, for example, PP2A and TH, or decrease activity upstream of Fyn tyrosine kinase are also within the scope of Fyn tyrosine kinase inhibitors of the present invention.
As used herein, the term “neurodegeneration” refers the damage or death of a cell in the central nervous system, for example, a neuron. Neurodegeneration refers to any pathological changes in neuronal cells, including, without limitation, death or loss of neuronal cells and any changes that precede cell death. The pathological changes may be spontaneous or may be induced by any event and include, for example, pathological changes associated with apoptosis. The neurons may be any neurons, including without limitation sensory, sympathetic, parasympathetic, or enteric, e.g. dorsal root ganglia neurons, motor neurons, and central neurons, e.g. neurons from the spinal cord.
As used herein, the terms “neurodegenerative disorder” or “neurodegenerative disease” refer broadly to disorders or diseases that affect the nervous system having damage or death of a cell of the central nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis.
As used herein, the term “compound” refers to a polynucleotide, a protein, a polypeptide, a peptide, an antibody, an immunoglobulin, a ligand, a cytokine, a growth factor, a nucleic acid, a lipid, membrane, a carbohydrate, a drug, a prodrug, or a small molecule or a fragment thereof.
As used herein, the term “modulates” refers to the ability of a compound to alter the mRNA or protein expression level or kinase activity or phosphatase activity of a protein.
As used herein, the term “pharmaceutically acceptable carrier” refers to any carrier, diluent, excipient, wetting agent, buffering agent, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant, or sweetener, preferably non-toxic, that would be suitable for use in a pharmaceutical composition.
As used herein, the terms “pharmaceutically effective” or “therapeutically effective” shall mean an amount of a Fyn tyrosine kinase inhibitor that is sufficient to show a meaningful patient benefit, i.e., treatment, prevention, amelioration, or a decrease in the frequency of the condition or symptom being treated. The Fyn tyrosine kinase inhibitor may be administered in the form of a pharmaceutical composition with a pharmaceutically acceptable carrier.
As used herein, the term “treating” refers to: (i) preventing a disease, disorder or condition from occurring in an animal or human that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and/or (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition. For example, with respect to Parkinson's disease, treatment may be measured by quantitatively or qualitatively to determine the presence/absence of the disease, or its progression or regression using, for example, symptoms associated with the disease or clinical indications associated with the pathology.
As used herein, the term “polypeptide” is interpreted to mean a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides. “Polypeptide(s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide(s)” refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. “Polypeptide(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well-known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADPribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-link formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626 646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48 62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
As used herein, the term “polynucleotide” is interpreted to mean a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “nucleic acid” typically refers to large polynucleotides. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces T.
Methods for Treating or Preventing Neuroinflammation
The present invention relates generally to compositions and methods for treating and preventing Fyn kinase-modulated disease in a subject in need thereof. The invention is useful, for example, for slowing, protecting from the effects of, or halting the progression of a neuroinflammatory disease. In certain embodiments, the disease or disorder contemplated within the invention is associated with pathological Fyn kinase.-mediating signaling. Non-limiting examples of Fyn kinase-modulated disease that are treatable or preventable with the compositions and methods of the present invention include, but are not limited to, Parkinson's Disease, Alzheimer's Disease (AD), amnestic mild cognitive impairment (MCI), Down syndrome dementia, traumatic brain injury, Lewy body dementia, fronto-temporal dementia, and after stroke aphasia. It should be noted that the present invention is not limited to a particular type of Fyn kinase-modulated disease. The subject may be an animal, preferably a mammal or human.
The present invention also provides a pharmaceutical composition for treating an Fyn kinase-modulated disease in a subject, wherein the composition comprises an inhibitor of Fyn receptor kinase activity and a carrier. For example, in certain embodiments, the composition comprises an isolated nucleic acid, isolated peptide, antibody, small molecule, antagonist, aptamer, or peptidomimetic that reduces the activity of Fyn.
In other embodiments, the present invention provides a composition for treating an Fyn kinase-modulated disease in a subject, wherein the composition comprises an inhibitor of Fyn tyrosine receptor kinase expression and a carrier. For example, in certain embodiments, the composition comprises an isolated nucleic acid (e.g., siRNA, ribozyme, antisense RNA, etc.) that reduces the expression level of Fyn receptor kinase in a cell.
In certain embodiments, the Fyn receptor kinase inhibitor is prepared as a prodrug. A prodrug is an agent converted into the parent drug in vivo. In one embodiment, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound. Prodrugs are known to those skilled in the art, and may be prepared using methodology described in the art. Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36Cl, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 18O, 32P, and 35S. In one embodiment, the isotope comprises deuterium. In certain embodiments, isotopically labeled compounds are useful in drug and/or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
In one embodiment, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
In one embodiment the inhibitor is a small molecule inhibitor of Fyn tyrosine selected from the group consisting of saracatinib, bosutinib, dasatinib, ponatinib, PP2, a pharmaceutically acceptable salt thereof, a derivative thereof, and any combinations thereof. In a preferred embodiment the small molecule inhibitor is saracatinib, otherwise known as AZD0530, or N-(5-chlorobenzo[d][1,3]dioxol-4-yl)-7-(2-(4-methyl piperazin-1-yl)ethoxy)-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine)-, or pharmaceutically acceptable salts, prodrugs, solvates or derivatives thereof.
Saracatinib (AZD530), has the following chemical formula:
N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(oxan-4-yloxy)quinazolin-1-amine and is commercially available through Astra Zeneca. The compound is currently used to treat advanced solid tumors in pancreatic, ovarian, prostate, osteosarcoma, melanoma, and colon cancer.
Thus the invention provides a pharmaceutical composition comprising an effective amount of saracatinib, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a carrier.
In some embodiments an effective amount can include an amount sufficient for a trough cerebral spinal fluid (CSF) concentration of saracatinib in the mammal of at least about 0.9 nM. In other embodiments, the trough CSF concentration of saracatinib in the mammal is at least about 2.1 nM. In yet other embodiments, the trough CSF concentration of saracatinib in the mammal is at least about 2.5 nM. In yet other embodiments, the trough CSF concentration of saracatinib in the mammal ranges from about 0.9 nM to about 2.2 nM. In yet other embodiments, the trough CSF concentration of saracatinib in the mammal ranges from about 2.1 nM to about 8.3 nM. In yet other embodiments, the trough CSF concentration of saracatinib in the mammal ranges from about 2.5 nM to about 14.0 nM. In yet other embodiments, the trough CSF concentration of saracatinib in the mammal ranges from about 0.9 nM to about 14.0 nM. In yet other embodiments, the average brain concentration of saracatinib in the mammal is selected from the group consisting of at least about 3 nM, at least about 7 nM, and at least about 8 nM. In yet other embodiments, the average brain concentration of saracatinib in the mammal ranges from about 3 to about 46 nM.
In certain embodiments, the saracatinib is saracatinib free base. In other embodiments, the saracatinib is saracatinib difumarate. In yet other embodiments, the saracatinib is selected from the group consisting of saracatinib free base, saracatinib difumarate, and any combinations thereof.
In certain embodiments, the methods and compositions further comprise administering to the mammal at least one additional or a second agent that treats or prevents Parkinson's or other neurological disease states. In yet other embodiments, the method further comprises administering to the mammal at least one additional agent that improves or prevents further neurodegeneration in the mammal. Pharmaceutical compositions of the invention can include an additional agent that is co-formulated with the Fyn tyrosine receptor inhibitor.
Examples of additional agents that treat or prevent Parkinson's disease or neurodegeneration and may be co-administered according to the invention include but are not limited to:
Carbidopa—currently the most effective Parkinson's disease medication. It is a natural chemical that is converted to dopamine;
Levodopa which is combined with carbidopa (Rytary, Sinemet), which protects levodopa from premature conversion to dopamine outside of the brain, which prevents or lessens side effects such as nausea;
Carbidopa-levodopa infusion, Duopa: carbidopa and levodopa which is administered through a feeding tube that delivers the medication in a gel form directly to the small intestine;
Dopamine agonists: Agents which mimic dopamine effects in the brain. Examples of dopamine antagonists include pramipexole (Mirapex), ropinirole (Requip) and rotigotine (given as a patch, Neupro). A short-acting injectable dopamine agonist, apomorphine (Apokyn), is used for quick relief;
MAO-B inhibitors: These medications include selegiline (Eldepryl, Zelapar) and rasagiline (Azilect). They help prevent the breakdown of brain dopamine by inhibiting the brain enzyme monoamine oxidase B (MAO-B). This enzyme metabolizes brain dopamine.
Catechol-O-methyltransferase (COMT) inhibitors. Entacapone (Comtan) is the primary medication from this class. This medication mildly prolongs the effect of levodopa therapy by blocking an enzyme that breaks down dopamine. Tolcapone (Tasmar) is another COMT inhibitor that is rarely prescribed due to a risk of liver damage anticholinergic medications such as benztropine (Cogentin) or trihexyphenidyl.
Amantadine. provides short-term relief of symptoms of mild, early-stage Parkinson's disease. It may also be given with carbidopa-levodopa therapy during the later stages of Parkinson's disease to control involuntary movements (dyskinesias) induced by carbidopa-levodopa.
In another aspect of the invention the Fyn kinase inhibitor may be combined with surgical procedures. One example is deep brain stimulation. In deep brain stimulation (DBS), surgeons implant electrodes into a specific part of the brain. The electrodes are connected to a generator implanted in the chest near the collarbone that sends electrical pulses to the brain and can reduce Parkinson's disease symptoms. DBS can stabilize medication fluctuations, reduce or halt involuntary movements (dyskinesias), reduce tremor, reduce rigidity, and improve slowing of movement.
In another aspect of the invention the Fyn tyrosine kinase inhibitor is a polynucleotide, polynucleotide that includes but is not limited to an antisense polynucleotide, ribozyme, RNA interference (RNAi) molecule, small hairpin RNA (shRNA), triple helix polynucleotide and the like, where the nucleotide sequence of such polynucleotides are the nucleotide sequences of DNA and/or RNA. Antisense technology may be used to achieve Fyn tyrosine kinase-specific interference, using for example, stoichiometric amounts of single-stranded nucleic acid complementary to the messenger RNA of Fyn tyrosine kinase which are introduced into the cell.
In one embodiment, an RNA interference (RNAi) molecule is used as a Fyn tyrosine kinase inhibitor, decreasing Fyn tyrosine kinase gene expression in a cell. In another aspect, the Fyn tyrosine kinase inhibitor is a siRNA molecule for targeting Fyn tyrosine kinase in a mammal, including without limitation, Fyn tyrosine kinase siRNA for a mouse, rat, monkey, or human.
In one embodiment, the compositions and methods of the present invention includes a Fyn tyrosine kinase inhibitor of at least one Fyn tyrosine kinase siRNA. In one aspect, the Fyn tyrosine kinase inhibitor includes a combination of differing Fyn tyrosine kinase siRNA molecules. Materials and methods to produce Fyn tyrosine kinase siRNA molecules are known in the art. The Fyn tyrosine kinase siRNA molecules may be produced by a number of methods, including the use of commercially available kits, for example, The SILENCER™ siRNA Construction Kit (Ambion, Austin, Tex.), a mammalian siRNA Fyn tyrosine kinase expression plasmid (Upstate Cell Signaling Solutions, Charlottesville, Va.), or obtained from MoleculA (Columbia, Md.).
The siRNA can be administered directly, for example, intracellularly, into a cell to mediate RNA interference (Elbashir et al., 2001, Nature 411:494 498) or administered extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be administered by contacting the cell with a solution containing the RNA. Physical methods of introducing polynucleotides, for example, injection directly into the cell or extracellular injection into the organism, may also be used. Other methods known in the art for introducing polynucleotides to cells may be used, such as viral vectors, viruses, lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, electroporation, the like. siRNA can be made using, for example, chemical synthesis or in vitro or in vivo transcription. A number of expression vectors have also been developed to continually express siRNAs in transiently and stably transfected mammalian cells (Brummelkamp et al., 2002 Science 296:550 553; Sui et al., 2002, PNAS 99(6):5515 5520; Paul et al., 2002, Nature Biotechnol. 20:505 508
In one embodiment, the Fyn tyrosine kinase inhibitor is a small hairpin RNA (shRNA), which is processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may also be useful for specific applications. The RNA molecule may be at least 10, 12, 15, 20, 21, 22, 23, 24, 25, 30, nucleotides in length.
RNA containing a polynucleotide sequence identical to a portion of Fyn tyrosine kinase gene is preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence of Fyn tyrosine kinase may also be effective for inhibition. Thus, one hundred percent sequence identity between the RNA and the target gene is not required to practice the present invention. Greater than 80% or 90% sequence identity or 100% sequence identity, between the inhibitory RNA and the portion of the Fyn tyrosine kinase is preferred. Thus, sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group).
Fyn tyrosine kinase inhibitors can be delivered intracellularly using a lipid-mediated protein delivery system. Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy A G, Caspase-3 dependent proteolytic activation of Fyn tyrosine kinasemediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci. 2003 September; 18(6):1387-401. The incomplete cleavage of PKCδ or PKCδ-mediated apoptotic inhibition can be measured using standard techniques known to one skilled in the art. Yoshimura S, Banno Y, Nakashima S, Takenaka K, Sakai H, Nishimura Y, Sakai N, Shimizu S, Eguchi Y, Tsujimoto Y, Nozawa Y. Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation. J Biol Chem. 1998 Mar. 20; 273(12):6921-7 (describing a method to determine caspase activity). Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy A G, Caspase-3 dependent proteolytic activation of Fyn tyrosine kinasemediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci. 2003 September; 18(6):1387-401) (describing the use of PKCδ-specific antibodies). Reyland M E, Anderson S M, Matassa A A, Barzen K A, Quissell D O. Fyn tyrosine kinase is essential for etoposide-induced apoptosis in salivary gland acinar cells. J Biol Chem. 1999 Jul. 2; 274(27):19115-23), Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy A G. Caspase-3-dependent proteolytic cleavage of protein kinase C delta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl. J Neurosci. 2002 Mar. 1; 22(5):1738-51, (describing assaying for Fyn tyrosine kinase enzymatic activity using an immunoprecipitation assay).
In one embodiment, the compositions and methods of the present invention include disruption of the Fyn-PKCδ pathway, particularly a PKCδ serine- and threonine kinase inhibitor or an RNAi molecule used as a PKCδ serine- and threonine kinase inhibitor. A suitable small molecule inhibitor of PKCδ serine- and threonine kinase can be selected from the group consisting of Balanol (n-tosyl derivative), δV1-1 (KAI-9803 or delcasertib), Rottlerin (5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chrmene), a pharmaceutically acceptable salt thereof, a derivative thereof, and any combinations thereof. Suitable methods for RNAi molecule design and delivery, targeted for inhibition of PKCδ serine- and threonine kinase, are synonymous with those describe above.
In another embodiment of the invention, other agents such as peptides, small molecules and the like may be screened to identify those that alter Fyn tyrosine kinase activity, particularly the ability of Fyn tyrosine kinase upregulation in response to inflammatory signals such as TNFα or LPB to identify additional Parkinson's disease treating agents. The Fyn tyrosine kinase inhibitor may directly alter the interaction of proteins in the Fyn tyrosine kinase pathway, for example, by inhibiting the activity of a kinase or phosphorylase in the pathway or by interfering with a step of the pathway.
The inhibition or reduction of Fyn tyrosine kinase activity can be determined using a variety of methods and assays routine to one skilled in the art, for example, determining the phosphorylation and/or activation of a Fyn tyrosine kinase kinase's target. Generally, a purified or partially purified Fyn tyrosine kinase is incubated with a peptide comprising the target sequence of Fyn tyrosine kinase under conditions suitable for the kinase to phosphorylate its target sequence of amino acids (i.e., protein, polypeptide). The particular requirements of the kinase may be determined empirically by one of skill in the art, or the conditions that have been published for a particular kinase may be used. The extent of phosphorylation of the target peptide is determined in the presence and absence of the test compound and may be determined in the presence of varying concentrations of the test compound. The phosphorylation rate may be determined by any means known in the art including electrophoretic assays, chromatographic assays, phosphocellulose assays and the like.
In an electrophoretic assay, a radiolabeled phosphate donor such as ATP or GTP is incubated with the peptide substrate in the presence of a kinase. The phosphorylated substrate versus the phosphate donor (e.g., ATP, GTP) is separated via thin-layer electrophoresis (Hunter J. Biol. Chem. 257:4843, 1982; incorporated herein by reference). Any matrix may be used in the electrophoresis step including polyacrylamide, cellulose, etc. The extent of phosphorylation may then be determined by autoradiography or scintillation counting.
The labeled phosphate donor may be separated from the phosphorylated amino acid sequence by standard chromatography techniques. Any matrix may be used to effect the separation including ion exchange resins, PEI cellulose, silica gel, etc. Standard column chromatography methods may be used, or HPLC methods may be used for faster cleaner separations. The radio-labeled peptides are detected by scintillation counting to determine the phosphorylation rate.
Another method which is historically the most popular is the phosphocellulose paper assay, first described by Witt et al. (Witt et al. Anal. Biochem. 66:253, 1975; incorporated herein by reference). Immunological methods may also be used to detect the phosphorylation of a peptide or protein substrate. For example, anti-phosphotyrosine or anti-phosphoserine antibodies may be used in the detection or precipitation of phosphorylated amino acid sequences. For example, multiple Fyn tyrosine kinase antibodies that detect the phosphorylated and unphosphorylated forms of Fyn tyrosine kinase are commercially available. (Cell signaling, Beverly, Mass. and Santa Cruz, Santa Cruz, Calif.).
In comparing the rates of phosphorylation in the presence and absence of the test compound, the compound should lead to at least a 10% decrease in the rate of phosphorylation, more preferably at least 25%, and most preferably at least 40%. These decreases are preferably obtained at micromolar concentrations of the compound and more preferably nanomolar concentrations (e.g., less than 100 nM).
In another aspect, the invention includes determining whether a potential Fyn tyrosine kinase inhibitor inhibits Fyn tyrosine kinase activity. The ability of a Fyn tyrosine kinase inhibitor to decrease Fyn tyrosine kinase activity, for example, neurodegeneration can be assessed using standard techniques known to one skilled in the art, including commercially available assays. These include apoptotic DNA Ladder assays, Cell Death Detection ELISAPLUS (from Roche Applied Sciences), Caspase-3 Activity AssayPLUS (from Roche Applied Sciences), terminal deoxynucleotidyl transferase-mediated dUTP [deoxy-uridine triphosphate] nick end labeling (TUNEL) assays. DNA-binding dyes or stains such as Hoechst 3342 or DAPI or propidium iodide may be used to assess nuclear morphology and DNA damage. Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy A G, Caspase-3 dependent proteolytic activation of Fyn tyrosine kinasemediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. Eur J Neurosci. 2003 September; 18(6): 1387-401.
In one aspect, the method includes administering a Fyn tyrosine kinase inhibitor. A Fyn tyrosine kinase inhibitor may be administered to an individual by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, a composition comprising a Fyn tyrosine kinase inhibitor can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant. A Fyn tyrosine kinase inhibitor also can be administered as a topical spray or an inhalant, in which case one component of the composition is an appropriate propellant.
The skilled artisan can readily perform the in vivo tests to determine, the amount or dose of Fyn tyrosine kinase inhibitor to administer, the formulation of the Fyn tyrosine kinase inhibitor, the route of administration of the Fyn tyrosine kinase inhibitor, and the time at which neurodegeneration or neuroprotection and dopamine synthesis or levels should be assessed.
Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma and cerebral spinal fluid may be measured, for example, by high performance liquid chromatography.
Neurodegeneration or neuroprotection can be detected using any number of methods. Histological, neurochemical and biochemical markers of dopamine producing cells. These techniques are routine and well-known to one skilled in the art. They include, for example, terminal deoxynucleotidyl transferase-mediated dUTP-X3′ nick end-labeling (TUNEL) assays that detect the free 3′ OH strand breaks resulting from DNA degradation which is associated with apoptosis (J Cell Biol 199: 493, 1992). In addition, kits that measure apoptotic cell death are also commercially available and include, for example, In Situ Cell Death Detection kit; Boehringer Mannheim, Mannheim or ApoTag, Oncor, Gaithersburg, Md.). Preparation of neuronal sections for apoptosis staining using the TUNEL technique is described in (Gorczyca, (1993) Cancer Res 53:1945-51). Apoptosis can also be detected using electrophoresis of the soluble DNA fraction isolated from neuronal cells by quantifying the ladder-like appearance as described in (PNAS 95: 2498, 1998) or using DNA binding dyes Hoechst 33342 and propidium iodide flow cytometry assay described in Dengler et al., (1995) Anticancer Drugs. 6:522-32.
The present invention also provides kits that can be used in the above methods. In one embodiment, a kit comprises at least one Fyn tyrosine kinase inhibitor, and optimally a second treating agent, in one or more containers, useful for the treatment of a disorder, disease, or condition associated with neurodegeneration or Parkinson's disease. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
All publications, patents and patent applications identified herein are incorporated by reference, as though set forth herein in full. The invention being thus described, it will be apparent to those skilled in the art that the same may be varied in many ways without departing from the spirit and scope of the invention. Such variations are included within the scope of the following claims.
The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention.
Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson's disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. While studying kinase signaling in PD models, Applicants unexpectedly discovered that the major non-receptor tyrosine kinase Fyn is a significant upstream mediator of microglial neuroinflammatory processes in PD. Applicants herein elucidate the role of Fyn in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPS- and TNFα-induced MAP kinase phosphorylation and activation of the NFκB pathway, implying that Fyn is a major upstream regulator of the proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn+/+) and Fyn knockout (Fyn−/−) mice revealed that Fyn is required for the pro-inflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn−/− and PKCδ−/− mice when compared to wild-type mice.
The pathophysiology of PD is complex and multifactorial, with mitochondrial dysfunction, oxidative stress, apoptosis and proteasomal dysfunction, being identified among others as potential disease mechanisms underlying nigrostriatal dopaminergic neuronal degeneration (Przedborski, 2005; Jenner and Olanow, 2006; Olanow, 2007; Levy et al., 2009). Recently, a wealth of data from cell culture, animal models and post-mortem analyses of human PD brains have established chronic, sustained microglia-mediated neuroinflammation as being a major event in the delayed and progressive loss of dopaminergic neurons within the SN (Imamura et al., 2003; Block et al., 2007; Glass et al., 2010; Tansey and Goldberg, 2010). As the macrophagic cells of the central nervous system (CNS), microglia compose a major component of the brain's innate immune system. Under ‘normal’ physiological conditions, they produce anti-inflammatory and neurotrophic factors to promote neuronal survival and plasticity (Carson, 2002). However, when they encounter a potential pathogen, a dead or dying neuron or neurotoxic stress, they switch to an ‘activated’ phenotype, producing pro-inflammatory cytokines and chemokines, reactive nitrogen species, and reactive oxygen species. Activated microglia may also directly contribute to cell death by phagocytizing dopaminergic neurons (Barcia et al., 2012; Virgone-Carlotta et al., 2013). Thus, the pathophysiology of PD is accompanied by a sustained pro-inflammatory microglial response that contributes to neuron death, thereby exacerbating disease progression.
Fyn, a member of the Src family of kinases, is a non-receptor tyrosine kinase expressed in the brain. The kinase has been shown to play a role in amyloid-mediated apoptosis in cortical neurons (Lambert et al., 1998), astrocyte migration (Dey et al., 2008) and oligodendrocyte differentiation (Sperber et al., 2001). In the peripheral immune system, Fyn plays a role in mast cell and B/T cell activation (Palacios and Weiss, 2004; Gomez et al., 2005a). Fyn was shown to mediate pro-inflammatory mediator production in mast cells, macrophages, basophils as well as in natural killer cells (Raj asekaran et al., 2013). Fyn was shown to be activated following fibrillar β-amyloid peptide engagement of its receptor CD36, contributing to activation and migration of primary murine peritoneal macrophages (Moore et al., 2002; Stuart et al., 2007), and in BV2 microglial cells stimulated with the neurotoxic fragment of prion protein (Kouadir et al., 2012). Recently, we have identified a pro-apoptotic Fyn/PKCδ-mediated signaling pathway that contributes to oxidative stress-induced cell death in dopaminergic neurons (Kaul et al., 2005; Saminathan et al., 2011). However, the role of Fyn in microglial activation and neuroinflammation has never been studied in PD. Therefore, Applicants sought to characterize the role of the Fyn-PKCδ signaling pathway in microglial activation and neuroinflammation in cell culture and animal models of PD. The results from these comprehensive studies reveal that Fyn kinase plays a key role in microglial activation and sustained neuroinflammation in the nigral dopaminergic system.
Primary mouse microglia were prepared as described in our recent publication using a magnetic separation method, which enables us to obtain a high-yield pure fraction of microglia from mixed glial cultures (Gordon et al., 2011). Iba-1 and GFAP immunocytochemistry confirmed that the microglial fraction obtained after magnetic separation was devoid of astrocytes (
Our initial experiment to determine whether the non-receptor tyrosine kinase Fyn plays a role in regulating neuroinflammatory responses in PD was carried out in BV2 microglial cells, which are widely used in vitro models of neuroinflammation (Henn et al., 2009; Gao et al., 2011; Kim et al., 2013b). We treated BV2 cells with 1 μg/mL LPS for 10-60 min and measured Fyn activity using an in vitro kinase assay (Saminathan et al., 2011). A kinase reaction mixture containing 32P-ATP and a Fyn-specific peptide substrate were added to whole cell lysates. LPS stimulation of BV2 microglia rapidly induced Fyn activity as early as 10 min post-LPS stimulation (
Next, we extended our studies to primary microglia derived from both wild-type and Fyn-deficient (Fyn−/−) mice. These were treated with 200 ng/mL LPS for 0-30 min. In line with the analyses of BV2 cells, stimulation of the primary microglia from Fyn+/+ mice rapidly increased the levels of p-Y416 SFK (
It has been shown that Src family kinases, including Fyn, phosphorylate PKCδ at residue Y311 in platelets and in immortalized dopaminergic neuronal cells (Steinberg, 2004; Saminathan et al., 2011). Therefore, we investigated if Fyn-PKCδ signaling regulates microglial pro-inflammatory responses using primary microglia cultures from wild-type, Fyn−/− and PKCδ−/− mice. Stimulation with LPS induced a rapid and time-dependent increase in p-Y311 PKCδ in wild-type microglia. In contrast, LPS failed to increase Y311 phosphorylation of PKCδ in the Fyn−/− microglia (
We next examined whether the Fyn-PKCδ signaling axis plays a role in mediating activation of the MAP kinase pathway, a key hallmark of neuroinflammatory signaling in microglia. MAP kinases are important regulators of pro-inflammatory cytokine synthesis in microglial cells (Koistinaho and Koistinaho, 2002; Tansey and Goldberg, 2010). For this purpose, we treated wild-type, Fyn−/−, and PKCδ−/− microglia with LPS for 15, 30 and 45 min each and determined MAPK activation. The LPS treatment significantly increased the phosphorylation of p38 and p44/42 (p-ERK) kinases in wild-type microglia (
Pro-inflammatory signaling mediated by both LPS and TNFα converges at the NFκB pathway. Activation of NFκB signaling during the pro-inflammatory process is characterized by the phosphorylation and subsequent degradation of the inhibitory protein IκBα, after which the NFκB p65-p50 heterodimer enters the nucleus, leading to the transcription of various pro-inflammatory genes (Hayden and Ghosh, 2004). To elucidate whether the Fyn mediates the nuclear translocation and activation of NFκB signaling in activated microglia, primary microglia obtained from wild-type and Fyn−/− microglia were treated with LPS for 15-45 min. Whole cell lysates were prepared and probed for IκBα. LPS treatment induced a greater degradation of IκBα in wild-type microglia than in Fyn−/− microglia at the 15 minute time point, followed by the resynthesis of IκBα 30 and 45 minutes post stimulation in the WT cells. Resynthesis of IκBα in Fyn−/− microglia was almost completely abrogated, indicating diminished NFκB activation (
Next, we determined whether Fyn-PKCδ signaling axis regulates microglia-mediated pro-inflammatory mediator production. After treating wild-type, PKCδ−/− and Fyn−/− microglial cultures with LPS or TNFα, we utilized multiplexed immunoassays to quantify inflammogen-induced cytokine secretion. We observed significant production of the cytokines IL-6, IL-12p70, and TNFα from wild-type microglia treated with LPS (
Fyn/PKCδ Regulates the Induction of Neuroinflammatory Markers iNOS and gp91phox in Microglia During LPS Stimulation
We further assessed whether Fyn alters the induction of iNOS and gp91phox, which are key pro-inflammatory responses of microglial activation following LPS treatment. Treatment with LPS induced a stronger nitrite response from wild-type microglia than from Fyn−/− microglia (
Fyn−/− and PKCδ−/− Mice are Resistant to LPS- and MPTP-Induced Neuroinflammatory Responses
To extend our findings from isolated primary microglia to in vivo animal models of neuroinflammation, we first used the LPS model, which has previously been used to evoke neuroinflammatory responses in vivo (Choi et al., 2007; Qin et al., 2007). Wild-type (PKCδ+/+ and Fyn+/+), PKCδ−/− and Fyn−/− mice were injected with 5 mg/kg LPS or PBS and were sacrificed 3 h later. Striatal mRNA contents of the pro-inflammatory cytokines pro-IL-1β and TNFα were determined by qRT-PCR. The levels of cytokine induction were almost identical in both wild-type groups, and we thus pooled the results. Systemic LPS administration strongly increased the levels of pro-IL-1β and TNFα transcripts in wild-type striata, but not in Fyn−/− and PKCδ−/− striata (
Fyn−/− and PKCδ−/− Mice are Protected Against 6-OHDA-Induced Nigrostriatal Dopaminergic Neuronal Deficits and Microgliosis
The 6-OHDA mouse model has recently been shown to elicit a neuroinflammatory response and neurodegeneration in the nigrostriatal dopaminergic system (Stott and Barker, 2014). While studying the role of Fyn in dopamine D1 receptor agonist-induced redistribution of NMDA receptor subunits, it was serendipitously discovered that Fyn−/− mice were remarkably intransigent to 6-OHDA-induced behavioral deficits and striatal TH loss (Dunah et al., 2004). Fyn+/+ and Fyn−/− mice, injected unilaterally with 6-OHDA (
In the next set of in vivo experiments, we checked whether PKCδ−/− mice were also resistant to 6-OHDA-induced nigral microgliosis and dopaminergic neuronal loss. PKCδ+/+ and PKCδ−/− mice were injected unilaterally with 6-OHDA for 9 days, and DAB-TH immunostaining was performed on striatal sections as described above (
Recently, it was demonstrated that treating mice with MPTP rapidly increased the number of microglial-neuronal appositions, termed gliapses (Barcia et al., 2012). These contacts preceded neuronal phagocytosis by the microglia. Similar appositions between microglia and dopaminergic neurons were demonstrated in the 6-OHDA model, with evidence suggesting that microglial cells actually phagocytized neurons (Virgone-Carlotta et al., 2013), which has been postulated to occur if the neurons are dysfunctional. Our confocal high magnification Z stack image analysis (Imaris software) revealed a sharply increased number of microglial-neuronal contacts formed in the Fyn+/+ SN post-6-OHDA treatment as indicated by arrowheads (
Thus far, our results demonstrated that short-term treatment of microglial cells with LPS and TNFα brings about an increase in Fyn activity, but not its expression. Strikingly, we discovered that prolonged treatment (12-24 h) of microglia with LPS or TNFα actually resulted in increased Fyn expression, evidenced by Western blot and immunocytochemistry (
Evidence from experimental models and human PD post-mortem studies strongly implicates the microglial-mediated inflammatory response as a major driver in the progression of PD; however, the key upstream cell signaling mechanisms that govern the neuroinflammatory processes have yet to be elucidated. Our results obtained from both cell culture and animal models provide novel insight into the role of the Fyn-PKCδ signaling cascade in regulating microglia-mediated neuroinflammation as related to PD pathogenesis. We have demonstrated dual regulation of pro-neuroinflammatory responses in microglia involving post-translational tyrosine phosphorylation of Fyn at its activation loop during the early stages of an inflammatory insult as well as transcriptional upregulation of Fyn upon prolonged exposure to pro-inflammatory stimuli. We have also showed that Fyn serves as a major upstream signaling molecule that works in concert with PKCδ to influence MAP kinase downstream and the NFκB pro-inflammatory cascade. Collectively, our study provides novel and significant insight into the pro-inflammatory function of Fyn-PKCδ signaling in PD models, and to the best of our knowledge, we are the first to discern this key signaling cascade that is relevant to microglia-mediated neuroinflammation in the nigrostriatal dopaminergic system.
We demonstrate that both the tyrosine kinase Fyn and the serine/threonine kinase PKCδ are differentially expressed in microglia and astrocytes (
Our group has previously shown that PKCδ kinase proteolytic activation promotes oxidative stress-induced pro-apoptotic signaling pathways in dopaminergic neuronal cells (Kaul et al., 2003; Zhang et al., 2007; Jin et al., 2011a; Jin et al., 2011b). Recently, it was demonstrated that PKCδ is proteolytically cleaved by caspase-3 in LPS-treated BV2 cells (Burguillos et al., 2011). In the present study, we demonstrate that activated Fyn associates with PKCδ to phosphorylate the Y311 site, resulting in increased PKCδ kinase activity (
MAP kinase activation is necessary for cytokine production in various immune cell types, including microglia (El Benna et al., 1996; Koistinaho and Koistinaho, 2002). We demonstrate that Fyn-PKCδ signaling contributes to MAP kinase phosphorylation during microglial activation. Both LPS and TNFα stimulations rapidly activated the p38 and p-ERK MAP kinases in WT, but to a significantly lesser extent in the Fyn−/− and PKCδ−/− microglia (
Classical activation of microglia by TLR and TNFR1 agonists produces pro-inflammatory cytokines and chemokines, which mediate the downstream effects of microglial activation. Recently, we showed that TNFα directly induces dopaminergic neuronal apoptosis (Gordon et al., 2012). In our present study, the induction of the cytokines IL-6, IL-12 and TNFα was all diminished in Fyn−/− and PKCδ−/−_microglia in comparison to wild-type microglia (
We extended our in vitro studies to well-characterized animal models of neuroinflammation, wherein a single intraperitoneal injection of LPS in mice increases TNFα in the brain to levels that remain elevated long after serum TNFα levels have returned to normal (Qin et al., 2007). We utilized this model system to check for LPS-induced striatal pro-inflammatory cytokine induction in wild-type, Fyn−/− and PKCδ−/− mice. Strikingly, a single injection of LPS strongly increased WT striatal TNFα and pro-IL-1β mRNA levels; however, the induction of these cytokines was greatly diminished in Fyn−/− and PKCδ−/− striata (
Recent imaging studies have demonstrated the formation of glial-neuronal contacts, called gliapses, formed between dopaminergic neurons and microglia that precede neuron loss in the MPTP model (Barcia et al., 2012; Barcia et al., 2013). To determine whether Fyn plays a role in microglial-dopaminergic neuron contact formation, we adopted the 6-OHDA mouse model. The formation of gliapses was described recently in the 6-OHDA model (Virgone-Carlotta et al., 2013). Our results from high magnification confocal analysis revealed the formation of gliapses was almost completely blocked in 6-OHDA injected Fyn−/− mice (
As summarized in
Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12), ascorbic acid, RPMI, fetal bovine serum (FBS), L-glutamine, Hoechst nuclear stain, penicillin, streptomycin and other cell culture reagents were purchased from Invitrogen (Gaithersburg, Md.). Recombinant TNFα was purchased from Peprotech (Rocky Hill, N.J.), and LPS (E. coli 0111:B4, Endotoxin content 6.6000000 EU/mg) and 6-OHDA were purchased from Sigma (St. Louis, Mo.). The mouse Fyn antibody was purchased from Thermo Scientific (Waltham, Mass.). Antibodies for rabbit Fyn, PKCδ, p-Y311 PKCδ, IκBα, Lamin-B, NOS2 (iNOS) and mouse Tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Antibodies against rabbit p-Src family kinase Y416 (p-Y416 SFK), native p65, p-p38 MAP kinase, native p38 MAP kinase, p-p44/42 MAP kinase (p-ERK) and native p44/42 MAP kinase (ERK) were purchased from Cell Signaling (Beverly, Mass.). The gp91phox antibody was purchased from BD Biosciences (San Jose, Calif.). The mouse GFAP antibody was purchased from Millipore (Billerica, Mass.). The TH antibody was purchased from Chemicon (Temecula, Calif.). Mouse M2 FLAG and β-actin antibodies, as well as the rabbit β-actin antibody were purchased from Sigma. Rabbit and goat Iba-1 antibodies were purchased from Wako Chemicals (Richmond, Va.) and Abcam (Cambridge, Mass.), respectively. The goat TNFα antibody was purchased from R&D Systems (Minneapolis, Minn.). 32P-ATP was purchased from Perkin Elmer (Boston, Mass.) and the histone substrate from Sigma. The Bradford protein assay kit was purchased from Bio-Rad Laboratories (Hercules, Calif.). FLAG-tagged human WT Fyn and Y417A mutant Fyn constructs were obtained as described previously (Kaspar and Jaiswal, 2011).
The Fyn−/− and PKCδ−/− mice used in these studies were bred in our animal facility. Fyn−/− mice were originally obtained from Dr. Dorit Ron's laboratory at the University of California, San Francisco and are available from Jackson Laboratory (stock number 002271). PKCδ−/− mice were obtained originally from Dr. Keiichi Nakayama's laboratory (Division of Cell Biology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan). Wild-type (Fyn+/+ and PKCδ+/+), PKCδ−/− and Fyn−/− mice were housed under standard conditions of constant temperature (22±1° C.), humidity (relative, 30%), and a 12-h light cycle with food and water provided ad libitum. Six- to eight-week-old male mice were used for all studies. The well-characterized acute MPTP mouse model of PD (Wu et al., 2003; Przedborski et al., 2004; Kim et al., 2007; Hu et al., 2008) was primarily used for neuroinflammation studies. The mice from the MPTP treatment group received 4 intraperitoneal (i.p.) injections of MPTP-HCl (18 mg/kg free-base) dissolved in saline at 2-h intervals. Mice were sacrificed 24 h after the last injection. The nigral neuroinflammatory response was also studied using the systemic LPS injection model (Qin et al., 2007), which induces chronic neuroinflammation and progressive dopaminergic degeneration in mice. A single injection of LPS (5 mg/kg, i.p.) was delivered to wild-type, Fyn−/− and PKCδ−/− mice. Mice were sacrificed 24 to 48 h later. Control groups for both MPTP and LPS received equivolume injections of saline. We injected 2 μL of 6-OHDA, diluted at a concentration of 5 μg/μL in 0.02% ascorbic acid, into the left striatum (0.2 μL/min) using the Angle 2 stereotaxic apparatus (Leica Biosystems, St. Louis, Mo.). The coordinates, relative to bregma were: 0.7 mm anteroposterior, 2 mm lateral, and 2.4 mm ventral. The contralateral side was either not injected or injected with 2 μL of 0.02% ascorbic acid diluted in sterile PBS as a negative control. All animal procedures were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC).
Primary microglial cultures were prepared from wild-type, Fyn−/− and PKCδ−/− postnatal day 1 (P1) mouse pups as described previously (Gordon et al., 2011). Briefly, mouse brains were harvested, meninges removed, and then placed in DMEM-F12 supplemented with 10% heat-inactivated FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine, 100 μM non-essential amino acids, and 2 mM sodium pyruvate. Brain tissues were then incubated in 0.25% Trypsin-EDTA for 30 min with gentle agitation. The trypsin reaction was stopped by adding double the volume of DMEM/F12 complete medium and then washing brain tissues three times. Tissues were then triturated gently to prepare a single cell suspension, which was then passed through a 70-μm nylon mesh cell strainer to remove tissue debris and aggregates. The cell suspension was then made up in DMEM/F12 complete medium and seeded into T-75 flasks, which were incubated in humidified 5% CO2 at 37° C. The medium was changed after five to six days and the mixed glial cells were grown to confluence. Microglial cells were separated from confluent mixed glial cultures by differential adherence and magnetic separation to >97% purity, and then were allowed to recover for 48 h after plating. Primary microglia were treated in DMEM/F12 complete medium containing 2% FBS. For signaling experiments, the protocol employed by Stuart (2007) was utilized with a small modification. For this, the primary microglial cells were kept in 2% DMEM/F12 complete medium for 5 h at 37° C. prior to treatment. The microglial cells were treated with 100-200 ng/mL LPS and 10-30 ng/mL TNFα for durations sampled at pre-specified time points. We selected the LPS doses used in this study based on previous studies in which stimulation of cultured primary mouse microglia with 100 and 200 ng/ml LPS resulted in significant microglial activation (Haynes et al., 2006; Crotti et al., 2014; Lee et al., 2014).
siRNAs and Transfections of Microglia
Transient transfections of primary microglia with Fyn promoter reporter were performed using Lipofectamine LTX & Plus Reagent according to the manufacturer's protocol. Primary microglia were plated at 0.75×106 cells/well in 12-well plates one day before transfection. We transiently transfected 3 μg of Fyn promoter construct. Cells were treated 24 h after transfection with or without 200 ng/mL of LPS for 12 h and then lysed. Luciferase activity was measured using a Dual-luciferase assay kit (Promega) on a Synergy 2 multi-mode microplate reader (BioTek). Firefly luciferase luminescence values were used to normalize Renilla luciferase luminescence values. The pre-designed, on-target plus SMART pool Fyn siRNA (a combination of four siRNAs, Cat. No. LQ-040112-00-0002) and scrambled siRNA (Cat. No. D-001210-03-05) were purchased from Dharmacon (Lafayette, Colo.). We carried out siRNA transfections in primary mouse microglial cells with Lipofectamine 3000 reagent according to the manufacturer's protocol. Briefly, primary microglia were plated at 2×106 cells/well in 6-well plates one day before transfection. For each well, 300 pmol of Fyn siRNA pool (75 pmol each) or an equal amount of scrambled siRNA mixed with 5 μl of Lipofectamine 3000 were added to the cells. Seventy-two hours after the initial transfection, cells were analyzed by Western blotting to confirm the extent of Fyn knockdown or treated with LPS (200 ng/mL) for 24 further hours, after which cytokine content was analyzed by Luminex bioassay.
Transfection of BV2 microglia with WT Fyn-FLAG, Y417A Fyn-FLAG and Empty Vector pcDNA3.1 constructs was performed by using the AMAXA Nucleofector Kit. Briefly, BV2 cells were resuspended in transfection buffer (Solution 1: 400 μM ATP-disodium (Sigma A7699), 600 μM MgCl2-6H2O in water; Solution 2: 100 μM KH2PO4, 20 μM NaHCO3, 5 μM glucose in water) to a final concentration of 3×106 cells per 100 μl and mixed with the respective vector; 5 μg of vector DNA was used per transfection.
Immunohistochemistry was performed on sections from the substantia nigra and other brain regions of interest as described previously (Jin et al., 2011b; Ghosh et al., 2013). Briefly, mice were anesthetized with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine and then perfused transcardially with freshly prepared 4% paraformaldehyde (PFA). Extracted brains were post-fixed in 4% PFA for 48 h and 30-μm sections were cut using a freezing microtome (Leica Microsystems). Antigen retrieval was performed in citrate buffer (10 mM sodium citrate, pH 8.5) for 30 min at 90° C. Sections were then washed several times in PBS and blocked with PBS containing 2% BSA, 0.2% Triton X-100 and 0.05% Tween 20 for 1 h at room temperature. Sections were then incubated with primary antibodies overnight at 4° C. and washed 7 times in PBS on a Belly Dancer Shaker (SPI supplies). The sections were incubated with Alexa dye-conjugated secondary antibodies for 75 min at room temperature and their cell nuclei were stained with Hoechst dye. Sections were mounted on slides using Prolong antifade gold mounting medium (Invitrogen) according to the manufacturer's instructions. Samples were visualized using an inverted fluorescence microscope (Nikon TE-2000U) and images were captured using a Spot digital camera (Diagnostic Instruments Inc).
Immunofluorescence studies in primary microglia were performed according to previously published protocols with some modifications (Gordon et al., 2011). Briefly, microglial cells were grown on poly-D-lysine-coated coverslips and treated 48 h later. At the end of treatments, cells were fixed with 4% PFA, washed in PBS and incubated in blocking buffer (PBS containing 2% BSA, 0.5% Triton X-100 and 0.05% Tween 20) for 1 h at room temperature. The coverslips were then incubated overnight at 4° C. with respective primary antibodies diluted in PBS containing 2% BSA. Samples were then washed several times in PBS and incubated with Alexa 488 and 555 dye-conjugated secondary antibodies. The nuclei were labeled with Hoechst stain (10 μg/mL) and coverslips were mounted with Fluoromount medium (Sigma Aldrich) on glass slides for visualization. Quantification of the number of microglial/astroglial cells obtained post-separation was accomplished using JACoP, a downloadable ImageJ plugin from Fabrice P. Cordelieres. Original Hoechst or antibody TIFF files were converted into 8-bit black-and-white images, and a colocalization image was generated. Counting of Hoechst-positive and Iba-1+Hoechst-positive cells was done using the Cell counter function of the default ‘Analyze’ plugin in ImageJ.
Confocal imaging was performed at the Iowa State University Microscopy Facility, using a Leica DMIRE2 confocal microscope with the 63× and 43× oil objectives and Leica Confocal Software. One optical series covered 11-13 optical slices of 0.5-μm thickness each. Microglial neuronal contact identification and quantification were performed by counting the number of colocalizations of the two markers, with TH marked red by anti 555 and Iba-1 marked green by anti 488 using the methodology described by Barcia and colleagues (Barcia et al., 2012). The Imaris software was used to analyze the Z stack images for contact identification. The surface reconstruction wizard in the Imaris software was used to make 3-D reconstructions of stacks for easier viewing of microglial-dopaminergic contacts and surface topology.
qRT-PCR
RNA isolation from primary microglial cells and brain tissue samples was performed using the Absolutely RNA Miniprep Kit, and then 1 μg total of isolated RNA was used for reverse transcription with the AffinityScript qPCR cDNA synthesis system (Agilent Technologies) according to the manufacturer's instructions. Quantitative SYBR Green PCR assays for gene expression were performed using the RT2 SYBR Green Master Mix with pre-validated primers (SABiosciences qPCR assay system). Catalog numbers of the primers were Fyn—PPM04015A, pro-IL1β—PPM03109E, TNFα—PPM03113G. The mouse 18S rRNA gene (catalog number—PPM57735E) was used as the housekeeping gene for normalization. For each primer, the amount of template providing maximum efficiency without inhibiting the PCR reaction was determined during initial optimization experiments. For all experiments, dissociation curves were generated to ensure a single peak was obtained at the right melting temperature without non-specific amplicons. The fold change in gene expression was determined by the ΔΔCt method using the threshold cycle (Ct) value for the housekeeping gene and the respective target gene of interest in each sample.
Brain tissue and microglial cell lysates were prepared using modified RIPA buffer and were normalized for equal amounts of protein using the Bradford protein assay kit. Equal amounts of protein (12 to 25 μg for cell lysates and 30-40 μg for tissue lysates) were loaded for each sample and separated on either 12% or 15% SDS-PAGE gels depending on the molecular weight of the target protein. After separation, proteins were transferred to a nitrocellulose membrane and the nonspecific binding sites were blocked for 1 h using a blocking buffer specifically formulated for fluorescent Western blotting (Rockland Immunochemicals). Membranes were then probed with the respective primary antibodies for 3 h at room temperature or overnight at 4° C. After incubation, the membranes were washed 7 times with PBS containing 0.05% Tween 20, and then Secondary IR-680-conjugated anti-mouse (1:10,000, goat anti-mouse, Molecular Probes) and IR-800 conjugated anti rabbit (1:10,000, goat anti-rabbit, Rockland) were used for antibody detection with the Odyssey IR imaging system (LiCor). Membranes were visualized on the Odyssey infrared imaging system. Antibodies for β-actin and Tubulin were used as loading controls.
We adopted an immunoprecipitation (IP) protocol with slight modifications from Gao and colleagues (Gao et al., 2011). Cell lysates were prepared in TNE buffer (10 mM Tris-HCl at pH 7.5, 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, and 1:100 protease inhibitor cocktail) and centrifuged at 17,400×g for 40 min at 4° C. The supernatant protein concentration was measured and normalized between samples. Approximately 50 μL of the sample containing 20 μg protein was used as input. For immunoprecipitation analysis, 1 mg of protein in 400 μL TNE buffer was used. Next, 10 μL (2 μg) of Fyn rabbit polyclonal antibody was added to the lysates, and the samples were set on an orbital shaker overnight at 4° C. The next day, protein G Sepharose beads were spun down at 17,400×g for 5 min and the ethanol supernatant was replaced with an equal volume of the lysis buffer. The Protein G Sepharose slurry was washed once and 50 μL was added to each sample. The samples were set on an orbital shaker overnight at 4° C. Protein G beads were collected by centrifugation at 2000×g for 5 min and were washed four times with TNE buffer. The bound proteins were eluted by boiling in 2× protein-loading dye for 5 min. Immunoblots were performed on 12% SDS-PAGE gels as described for Western blotting.
Nuclear and cytoplasmic fractions were performed using the NE-PER Kit (Thermo Scientific) as previously described (Jin et al., 2011a; Jin et al., 2014). Briefly, 5×106 cells were treated with LPS or TNFα for 15 min. CER1 reagent (200 μL) was used for each sample to extract the cytoplasmic fraction, and 50 μL of NER reagent was used to extract the nuclear fraction. Tubulin or β-actin was used as a cytosolic fraction marker. Lamin B was used as a nuclear fraction marker.
Cell pellets were washed with ice-cold PBS and resuspended in lysis buffer (25 mM HEPES at pH 7.5, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1% Triton X-100, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 mM NaF, and 4 μg/mL each of aprotinin and leupeptin) (Kaul et al., 2005). Next, 50 μg of crude protein was incubated with 150 mM Fyn kinase substrate (Biomol), 100 mCi of [γ-32P] ATP, Src-Mn-ATP cocktail and Src reaction buffer (Millipore) for 10 min at 30° C. with agitation. To precipitate the Fyn kinase substrate peptide, 20 mL of 40% trichloroacetic acid was added. Then 25 μL of the mixture was spotted onto a P81 phosphocellulose square, and 5 min after spotting, the squares were washed five times in 0.75% phosphoric acid in PBS with a final wash step in acetone. The squares were transferred into a scintillation vial and the CPMA counts were read in a liquid scintillation system after adding 5 mL of scintillation cocktail to each vial.
PKCδ IP kinase activity assays were performed as described previously (Anantharam et al., 2002; Latchoumycandane et al., 2011; Harischandra et al., 2014) with some modifications for microglial cells. Briefly, primary microglial cells were collected after treatments, washed in ice-cold PBS and resuspended in a mild RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Pierce Biotechnology). The cells were placed on ice for 20 min to allow for complete lysis and then centrifuged at 16,200×g for 45 min. The supernatant protein concentration was determined using the Bradford protein assay kit. Samples were normalized to a uniform total protein concentration of 2 μg/mL, and then 200 μg of total protein in a 250 μL reaction volume was immunoprecipitated overnight at 4° C. using 5 μg of PKCδ antibody. The next day, protein-A agarose beads (Sigma-Aldrich) were incubated for 1 h at room temperature. The protein A-bound antibody complexes were collected and washed 3 times in 2× kinase assay buffer (40 mM Tris, pH 7.4, 20 mM MgCl2, 20 μM ATP, and 2.5 mM CaCl2), and then resuspended in the same buffer. The kinase reaction was started by adding 40 μL of the reaction buffer containing 0.4 mg of histone H1, 50 μg/mL phosphatidylserine, 4 μM dioleoylglycerol, and 10 μCi of [γ-32P] ATP at 3000 Ci/mM to the immunoprecipitated samples. The samples were then incubated for 10 min at 30° C. and the kinase reaction was stopped by adding 2×SDS loading buffer and boiling for 5 min. Proteins were separated on a 15% SDS-PAGE gel and the phosphorylated histone bands were imaged using a Fujifilm FLA 5000 imager. Image analysis and band quantification were performed using ImageJ.
Nitric oxide production by primary microglia was measured indirectly by quantification of nitrite in the supernatant using the Griess reagent (Sigma Aldrich). Microglia were plated in poly-D-lysine-coated 96-well plates at 1×105 cells/well. Cells were treated with 100 ng/mL of LPS for 24 h and after 100 μL of supernatant was collected from each well, an equal volume of the Griess reagent was added. The samples were incubated on a plate shaker at room temperature for 15 min until a stable color was obtained. The absorbance at 540 nm was measured using a Synergy 2 multi-mode microplate reader (BioTek Instruments) and the nitrite concentration was determined from a sodium nitrite standard curve.
Primary microglia obtained from wild-type, PKCδ−/− and Fyn−/− mice were seeded in poly-D-lysine-coated 96-well plates at 1×105 cells/well. The cells were treated for 24 h with 100-200 ng/mL LPS or 10 ng/mL TNFα. After treatment, 50 μL of supernatant from each well was collected and frozen at −80° C. The levels of cytokines and chemokines in the supernatants were determined using the Luminex bead-based immunoassay platform (Vignali 2000) and pre-validated multiplex kits (Milliplex mouse cytokine panel Millipore) according to the manufacturer's instructions.
Iba-1 diaminobenzidine (DAB) immunostaining was performed on striatal and substantia nigral sections as described previously (Ghosh et al., 2010). Briefly, mice were perfused with 4% PFA and brains were post-fixed with PFA for 48 h before storage in 30% sucrose. Fixed brains were embedded in O.C.T compound (Tissue-Tek) and stored frozen at −80° C. until the frozen blocks were sliced into 30-μm coronal sections using a cryostat. Sections were probed with the primary antibodies overnight at 4° C. and then incubated with biotinylated anti-rabbit secondary antibody. The sections were then treated with Avidin peroxidase (Vectastain ABC Elite kit). The DAB reagent was used for producing the brown colored stain. Grading of microglial morphology was performed as described elsewhere (Lastres-Becker et al., 2012). For microglial grading, images were sharpened in ImageJ so the morphology could be more clearly visualized. The cell counter function in the ‘Analyze’ plugin was used to count the number of Type A, B, C and D microglia in the ventral midbrain sections.
Data analysis was performed using Prism 4.0 (GraphPad Software, San Diego, Calif.). The data were initially analyzed using one-way ANOVA and Bonferroni's post-test to compare the means of treatment groups. Differences of p<0.05 were considered statistically significant. Student's t-test was used when comparing two groups.
Neuroinflammation has been implicated as a major pathophysiological process of Parkinson's disease (PD) in recent years. Among various neuroinflammatory triggers, protein aggregates have been shown to be a predominant pathological trigger for microglial activation and subsequent proinflammatory cytokine and chemokine production in the brain, which in turn contributes to the accelerated progression of neurodegenerative processes. Also, emerging evidence indicates that aggregated pathogenic proteins, including α-synuclein (αSyn), are packaged into exosomes, which propagate protein aggregates from affected neurons to other brain cells, including microglial cells, through a non-cellular autonomous process, leading to a heightened neuroinflammatory response. Despite these advances, the cellular mechanisms underlying microglia-mediated neuroinflammatory events following stimulation with αSyn aggregates and αSyn-containing exosomes are yet to be defined.
Although endogenous αSyn protein is involved in many synaptic functions, misfolded αSyn serves as an endogenous antigen, provoking a self-propelling cycle of glial activity and upregulated proinflammatory mediators under both in vitro and in vivo conditions (1, 7-10). Emerging evidence indicates that aggregated extracellular αSyn can spread via a prion-like transmission mechanism, thereby spreading αSyn pathology to neighboring neuronal and glial cells (2, 3, 8, 11-14). Though its secretory mechanisms remain controversial, the exosomal release of pathogenic αSyn has been emerging as a novel concept in CNS diseases progression (2, 4, 5, 15). Also, αSyn aggregates are known to interact with microglial immune receptors. Recently, Kim et al. provided evidence that TLR2, but not TLR3 or TLR4, acts as a receptor for extracellular αSyn in microglia (16). Additionally, the pattern recognition receptor CD36 is involved in αSyn-mediated microglial activation (17). Thus, αSyn aggregates can activate TLR2/CD36 receptors. Nevertheless, the key signaling pathways involved in aggregated αSyn-mediated neuroinflammation in the nigral dopaminergic system are largely unknown. This idea of extracellular αSyn as an immunogenic agent to glial cells is extremely appealing given its emerging role in disease progression. Hence, the novel goal of this study is to characterize the role of a major non-receptor tyrosine kinase, Fyn, in regulating αSyn aggregate-induced neuroinflammation in PD. Apart from providing novel insights into the signaling mechanisms mediating neuroinflammatory responses during protein aggregation in PD pathology, this study also provides translational potential for developing neuroprotective strategies targeting Fyn kinase signaling during neuroinflammation in PD related neurodegenerative diseases.
Aggregated Human α-Synuclein Acts as an Efficient Danger Signal for the NLRP3 Inflammasome, Inducing IL-1β Maturation and Release
Misfolded proteins can activate the NLRP3 inflammasomes in microglia (Hafner-Bratkovic et al., 2012; Halle et al., 2008). After obtaining and purifying human αSyn (
The first step in the activation process of the NLRP3 inflammasome is priming, which entails the NF-κB-p65-mediated induction of pro-IL-1β and NLRP3, which occurs subsequent to the engagement of appropriate pro-inflammatory ligands or inflammogens to their respective receptors (Hayden and Ghosh, 2004). We demonstrated in Example 1 how inflammogen stimulation of microglia resulted in rapid Fyn activation, and Fyn-mediated PKCδ tyrosine phosphorylation and activation, which contributed to LPS- and TNFα-mediated cytokine production and pro-inflammatory signaling (Panicker et al., 2015). Accordingly, we primed WT and Fyn−/− microglia with various doses of LPS and TNFα to test the role of Fyn in NLRP3 inflammasome priming as assessed by blotting for pro-IL-1β and NLRP3. As expected, LPS and TNFα elicited a dose-dependent increase of pro-IL-1β and NLRP3 levels in WT microglia in contrast to a significantly lower response in Fyn−/− microglia (
The current model of the NLRP3 inflammasome postulates a two-step mechanism. A TLR or TNFR1 agonist, acting as the initial signal, induces the expression of pro-IL-13 and NLRP3 proteins. Subsequently, a second signal causes lysosomal rupture, mitochondrial ROS generation and/or K+ efflux that acts as a danger signal resulting in the assembly and activation of the inflammasome complex and the processing and secretion of IL-1β into the cell supernatant. Since we observed that αSyn can signal the inflammasome and can also amplify LPS-mediated activation of NLRP3 and pro-IL-1β at the mRNA and protein level, we wondered if it could activate the NLRP3 inflammasome independent of an LPS-mediated priming step. Unprimed and immortalized WT and Capsase-1−/− microglial cells were treated with aggregated αSyn for 4 h. As assessed via immunoblot, aggregated αSyn induced pro-IL-1β and NLRP3 to equivalent levels in both cell types (
We then looked for the signaling mechanism through which αSyn activates the NLRP3 inflammasome in microglia. Various studies have indirectly implicated disparate receptors in binding to and mediating αSyn signaling. Kim et al. (2013) considered TLR2 essential for αSyn-induced pro-inflammatory signaling and to contribute to αSyn import into microglia. TLR4-, CD36- and FcγR-deficient microglia have demonstrated attenuated neuroinflammatory responses to αSyn treatment (Fellner et al., 2013). Misfolded αSyn also interacts with microglial TLR1/2 and mediates Myd-88-dependent pro-inflammatory signaling (Daniele et al., 2015). Since we had previously demonstrated early inflammogen-mediated activation of Fyn and PKCδ, we sought to link αSyn recognition by a microglial receptor to Fyn activation, which is known to occur downstream of CD36 (Chen et al., 2008; Moore et al., 2002) and TLR2 (Finberg et al., 2012). We immunoprecipitated CD36 and TLR2 in control and αSyn-stimulated WT microglia and discovered that both CD36 and TLR2 interacted with αSyn, but only CD36 interacted with Fyn in αSyn-stimulated microglia (
Priming of the NLRP3 inflammasome involves activation of the NF-κB pathway downstream of TLR/TNFR1 engagement of their respective ligands. The NF-κB heterotrimeric complex comprises IκBα, p65 and p50. Upon inflammogen stimulation, IκBα is phosphorylated and rapidly degraded, allowing nuclear entry of the p65-NF-κB subunit. The transcription factor p65 binds to the promoters of various pro-inflammatory cytokine genes as well as the NLRP3 gene, bringing about their transcription, thereby leading to the production of pro-inflammatory cytokines/pre-cytokines. We have previously shown that Fyn contributes to LPS and TNFα-mediated NF-κB activation in microglia (Example 1; Panicker et al., 2015). To assess the role of Fyn in αSyn-mediated priming of the NLRP3 inflammasome, we first treated WT and Fyn−/− microglia with aggregated αSyn for 30 min, and then prepared nuclear and cytosolic extracts. Probing the nuclear extracts revealed less αSyn-induced nuclear translocation of p65 in the Fyn−/− microglial nuclear lysates relative to WT (
Fyn Contributes to αSyn Import into Microglial Cells, Thereby Contributing to NLRP3 Inflammasome-Associated Mitochondrial Dysfunction.
Although most existing studies agree on the steps to priming the NLRP3 inflammasome, the exact nature of the danger or activating signal remains contested. The current consensus points to lysosomal dysfunction, mitoROS generation and potassium efflux as possible molecular events leading to the assembly of the NLRP3 inflammasome. Since we observed Fyn interacting with CD36, a receptor protein involved in the uptake and aggregation seeding ability of amyloid (Sheedy et al., 2013), we tested whether Fyn plays a role in the uptake of αSyn into microglial cells by adding human αSyn to WT and Fyn−/− microglia for various time points. Cells were triple washed with PBS, then fixed and stained for microglial uptake of human αSyn, which showed up as intracellular puncta. The number of puncta per cell per field were quantified. Taken together, Fyn−/− microglia display diminished uptake of human αSyn (
Activation of the NLRP3 inflammasome in PD models is still an active area of investigation and has not been conclusively demonstrated yet. However, there is some evidence to suggest that it may be activated under certain conditions. Subjecting the transgenic db/db diabetic cell line to a regimen of the Parkinsonian toxicant MPTP activates the NLRP3 inflammasome and exacerbates neuroinflammation and neurodegeneration (Wang et al., 2014). Moreover, αSyn AAV overexpression in the nigra significantly increases IL-1β and TNFα in a rat model of synucleinopathy (Chung et al., 2009). Viral vector-mediated overexpression of IL-1β in the mouse SN can affect dopaminergic neurodegeneration (Ferrari et al., 2006). A recent study showed that NLRP3−/− mice were resistant to MPTP-induced TH-positive cell loss and dopaminergic neuron loss (Yan et al., 2015). Accordingly, to assess whether microglial inflammasome activation could be driven by αSyn overexpression in vivo we injected WT and Fyn−/− mice SNs with AAVs coding for GFP and human αSyn (
In this study microglial cell line were pretreated with 1-30 μM Saracatinib, a Fyn kinase inhibitor followed by aggregated α-synuclein (1 μM) for 24 hours in a 96 well plate. As shown in
Increasing evidences suggest that activated microglial cells in the SN pars compacta (SNpc) play an important role in dopaminergic neurodegeneration in human PD patients and in the MPTP mouse model. Thus, we evaluated whether the Sarcanatib attenuates microglial activation in the substantia nigra. C57 black mice were orally administered saracatinib (5 mg/kg, once) on days 1 and 2. On day 2, after sarcanatib administration, mice were subject to acute MPTP regimen. Mice were administered 18 mg/kg MPTP (4 i.p. injections at 2 hour intervals) and control animals received saline. Mice were sacrificed 24 hrs post last MPTP injection. Brain harvested and nigral tissue lysates were subject to Western blot analysis. As shown in
This invention was made with government support under contract NIH Grant No. R01 NS088206. The Government has certain rights in this invention.
