Patent Application
20110312894
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Nicotinic acetylcholine receptors (nAChRs) in mammals exist as a diverse family of channels composed of different, pentameric combinations of subunits derived from at least sixteen genes (Lukas et al., 1999; Jensen et al., 2005). Functional nAChRs can be assembled as either heteromers containing and β subunits or as homomers containing only
subunits (Lukas et al., 1999; Jensen et al., 2005). In the mammalian brain, the most abundant forms of nAChRs are heteromeric α4β2-nAChRs and homomeric α7-nAChRs (Whiting et al., 1987; Flores et al., 1992; Gopalakrishnan et al., 1996; Lindstrom, 1996; Lindstrom et al., 1996). α7-nAChRs appear to play roles in the development, differentiation, and pathophysiology of the nervous system (Liu et al., 2007b; Mudo et al., 2007).
nAChRs have been implicated in Alzheimer's disease (AD), in part because significant losses in radioligand binding sites corresponding to nAChRs have been consistently observed at autopsy in a number of neocortical areas and in the hippocampi of patients with AD (Burghaus et al., 2000; Nordberg, 2001). Attenuation of cholinergic signaling is known to impair memory, and nicotine exposure improves cognitive function in AD patients (Levin and Rezvani, 2002). In addition, several studies have suggested that the activation of α7-nAChR function alleviates amyloid-β (Aβ) toxicity. For instance, stimulation of α7-nAChRs inhibits amyloid plaque formation in vitro and in vivo (Geerts, 2005), activates α-secretase cleavage of amyloid precursor protein (APP) (Lahiri et al., 2002), increases acetylcholine (ACh) release and facilitates Aβ internalization (Nagele et al., 2002), inhibits activity of the MAPKINF-kB/c-myc signaling pathway (Liu et al., 2007a), and reduces Aβ production and attenuates tau phosphorylation (Sadot et al., 1996). These findings suggest that cholinergic signaling, mediated through α7-nAChRs, not only is involved in cognitive function, but also could protect against a wide variety of insults associated with AD (Sivaprakasam, 2006). Conversely, impairment of α7-nAChR-mediated cholinergic signaling during the early stage(s) of AD might play a pivotal role in AD pathophysiology.
In rat basal forebrain cholinergic neurons, α7 and β2 are the predominant nAChR subunits, and they were found to co-localize (Azam et al., 2003). Thus far, there has been no evidence that α7 and β2 subunits co-assemble to form functional nAChRs naturally, although functional α7β2-nAChRs have been reported using a heterologous expression system (Khiroug et al., 2002). As described herein, however, the inventors demonstrate that heteromeric α7β2-nAChRs exist in rodent basal forebrain cholinergic neurons and have high sensitivity to Aβ. There is a need in the art for a greater understanding of the role of nAChRs in learning and memory disorders, specifically Alzheimer's Disease, both in their functional characterization as well as the development of novel treatments for Alzheimer's Disease.
Various embodiments include a method of treating a neurodegenerative disorder in an individual, comprising providing a composition capable of inhibiting dysfunctional signaling of α7 nicotinic acetylcholine receptors (nAChRs), and administering a therapeutically effective amount of the composition to inhibit dysfunctional signaling of α7 nAChRs to treat the neurodegenerative disorder. In another method, the α7 nAChRs comprise heteromeric α7β2 nAChRs. In another embodiment, the composition comprises a β2 nAChR antagonist. In another embodiment, the neurodegenerative disorder comprises Alzheimer's Disease, dementia and/or epilepsy. In another embodiment, the neurodegenerative disorder comprises an early stage form of Alzheimer's Disease. In another embodiment, the composition comprises an α7 nAChR antagonist. In another embodiment, the composition comprises a therapeutically effective amount of compound comprising kynurenic acid (KYNA), methyllycaconitine (MLA), α-bungarotoxin (BGT), cholinesterase inhibitor, memantine, and/or α-conotoxin, or a pharmaceutical equivalent, derivative, analog and/or salt thereof. In another embodiment, inhibiting the dysfunctional signaling of α7 nAChRs comprises restoring function of heteromeric α7β2 nAChRs. In another embodiment, inhibiting the dysfunctional signaling of α7 nAChRs comprises protecting heteromeric α7β2 nAChRs from amyloid β (Aβ) effects. In another embodiment, the individual is a human. In another embodiment, the individual is a rodent. In another embodiment, the dysfunctional signaling of α7 nAChRs occurs in the brain medial septum and/or diagonal band in the individual.
Other embodiments include a method of diagnosing a neurodegenerative disorder in an individual, comprising obtaining a sample from the individual, assaying the sample to determine the presence or absence of dysfunctional signaling of α7 nicotinic acetylcholine receptors (nAChRs) in the individual, and diagnosing the neurodegenerative disorder based on the presence of dysfunctional signaling of α7 nAChRs in the individual. In another embodiment, the α7 nAChRs comprise heteromeric α7β2 nAChRs. In another embodiment, the individual is a human. In another embodiment, the individual is a rodent. In another embodiment, the neurodegenerative disorder comprises Alzheimer's Disease, dementia and/or epilepsy. In another embodiment, the dysfunctional signaling of α7 nAChRs occurs in the brain medial septum and/or diagonal band in the individual. In another embodiment, the neurodegenerative disorder has proven non responsive to treatment with galantamine, or a pharmaceutical equivalent, derivative, analog and/or salt thereof. In another embodiment, prior to obtaining the sample the individual is suspected of having a neurodegenerative disorder.
Various embodiments include a method of prognosing the onset of Alzheimer's Disease and/or dementia in an individual, comprising obtaining a sample from the individual, assaying the sample to determine the presence or absence of dysfunctional signaling of α7 nicotinic acetylcholine receptors (nAChRs) in the individual, and prognosing the onset of Alzheimer's Disease and/or dementia based on the presence of dysfunctional signaling of α7 nAChRs in the individual. In another embodiment, the α7 nAChRs comprise heteromeric α7β2 nAChRs. In another embodiment, the dysfunctional signaling of α7 nAChRs occurs in the brain medial septum and/or diagonal band in the individual.
Other embodiments include a method of diagnosing an increased likelihood of developing a neurodegenerative disorder relative to a normal subject in an individual, comprising obtaining a sample from the individual, assaying the sample to determine the presence or absence of dysfunctional signaling of α7 nicotinic acetylcholine receptors (nAChRs) in the individual, and diagnosing an increased likelihood of developing the neurodegenerative disorder relative to a normal subject based on the presence of dysfunctional signaling of α7 nAChRs in the individual. In another embodiment, the α7 nAChRs comprise heteromeric α7β2 nAChRs. In another embodiment, the neurodegenerative disorder comprises Alzheimer's Disease, dementia and/or epilepsy. In another embodiment, prior to obtaining the sample the individual is suspected of having a neurodegenerative disorder.
Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.
As used herein, the term. “Aβ” refers to amyloid beta peptides.
As used herein, the term “nAChR” refers to nicotinic acetylcholine receptor.
As used herein, the term “Aβ1-42” refers to amyloid beta peptides at positions 1-42 of the amyloid precursor protein (APP).
As used herein, the term “MS/DB” means medial septum/diagonal band.
As used herein, the term “AD” means Alzheimer's Disease.
As used herein, the term “dysfunctional signaling” refers to signaling mechanisms that are considered to be abnormal and not ordinarily found in a healthy subject or typically found in a population examined as a whole with an average amount of incidence.
As used herein, “treatment” or “treating” should be understood to include any indicia of success in the treatment, alleviation or amelioration of an injury, pathology or condition. This may include parameters such as abatement, remission, diminishing of symptoms, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating; improving a patient's physical or mental well-being; or, in some situations, preventing the onset of disease.
As used herein, “diagnose” or “diagnosis” refers to determining the nature or the identity of a condition or disease. A diagnosis may be accompanied by a determination as to the severity of the disease.
As used herein, “prognostic” or “prognosis” refers to predicting the outcome or prognosis of a disease.
As disclosed herein, nicotinic acetylcholine receptors (nAChRs) containing α7 subunits are believed to assemble as homomers. α7-nAChR function has been implicated in learning and memory, and alterations of α7-nAChR have been found in patients with Alzheimer's disease (AD). Findings in rodent, basal forebrain holinergic neurons are described herein consistent with a novel, naturally occurring nAChR subtype. In these cells, α7 subunits are coexpressed, colocalize, and coassemble with β2 subunit(s). Compared with homomeric α7-nAChRs from ventral tegmental area neurons, functional, heteromeric α7β2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the β2 subunit-containing nAChR-selective antagonist, dihydro-β-erythroidine (DH βE). Interestingly, heteromeric α7β2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid β1-42 (Aβ1-42). Slow whole-cell current kinetics, sensitivity to DHβE, and specific antagonism by oligomeric Aβ1-42 also are characteristics of heteromeric α7β2-nAChRs, but not of homomeric α7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Aβ when elicited from MS/DB neurons derived from nAChR β2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric α7β2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Aβ1-42 supports the existence of mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and could be targeted by disease therapies.
In one embodiment, the present invention provides a method of diagnosing susceptibility to a learning and/or memory disorder by determining the presence or absence of dysfunctional signaling of α7 containing nAChRs in a subject, where the presence of dysfunctional signaling of α7 containing nAChRs is indicative of susceptibility to the learning and/or memory disorder. In another embodiment, the α7 containing nAChRs comprise heteromeric α7β2-nAChRs. In another embodiment, the learning and/or memory disorder is Alzheimer's Disease. In another embodiment, the α7 containing nAChRs are found in basal forebrain cholinergic neurons. In another embodiment, the subject is a rodent. In another embodiment, the subject is a human.
In another embodiment, the present invention provides a method of diagnosing a learning and/or memory disorder by determining the presence or absence of dysfunctional signaling of α7 containing nAChRs in a subject, where the presence of dysfunctional signaling of α7 containing nAChRs is indicative of the learning and/or memory disorder. In another embodiment, the α7 containing nAChRs comprise heteromeric α7β2-nAChRs. In another embodiment, the learning and/or memory disorder is Alzheimer's Disease. In another embodiment, the α7 containing nAChRs are found in basal forebrain cholinergic neurons. In another embodiment, the subject is a rodent. In another embodiment, the subject is a human.
In one embodiment, the present invention provides a method of treating a learning and/or memory disorder in a subject by determining the presence of dysfunctional signaling of α7 containing nAChRs and inhibiting the dysfunctional signaling of α7 containing nAChRs. In another embodiment, the learning and/or memory disorder is Alzheimer's Disease. In another embodiment, inhibiting dysfunctional signaling of α7 containing nAChRs includes inhibiting expression of the nAChR α7 subunit. In another embodiment, inhibiting heteromeric α7β2-nAChR dysfunctional signaling includes the inhibition of expression of the nAChR β2 subunit. In another embodiment, the inhibition of expression of the nAChR β2 subunit includes fast whole-cell kinetics and/or low sensitivity to amyloid beta peptides.
As readily apparent to one of skill in the art, any number of readily available materials and known methods may be used to inhibit or activate nAChR signaling. For example, α7 nAChR antagonists such as α-conotoxin analogs (Armishaw, et al, Journal of Biological Chemistry, Vol. 285, No. 3; Armishaw, et al., Journal of Biological Chemistry, Vol. 284 No. 14), memantine (Aracava, et al., Journal of Pharmacology and Experimental Therapeutics, Vol. 312, No. 3), and kynurenic acid (Hilmas, et al., Journal of Neuroscience, 21(19): 7463-7473), may be used in conjunction with various embodiments herein to inhibit signaling of α7 containing nAChRs.
In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of compound that results in the inhibition of dysfunctional signaling of nAChRs. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, on as lyophilized powders.
The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins Pa., USA) (2000).
Typical dosages of an effective composition that results in the inhibition of dysfunctional signaling of nAChRs can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
Nicotinic acetylcholine receptors (nAChRs) containing α7 subunits are believed to assemble as homomers. α7-nAChR function has been implicated in learning and memory, and alterations of α7-nAChR have been found in patients with Alzheimer's disease (AD). Findings in rodent, basal forebrain holinergic neurons are described herein consistent with a novel, naturally occurring nAChR subtype. In these cells, α7 subunits are coexpressed, colocalize, and coassemble with β2 subunit(s). Compared with homomeric α7-nAChRs from ventral tegmental area neurons, functional, heteromeric α7β2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the β2 subunit-containing nAChR-selective antagonist, dihydro-P-erythroidine (DH βE). Interestingly, heteromeric α7β2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid β1-42 (Aβ1-42). Slow whole-cell current kinetics, sensitivity to DHβE, and specific antagonism by oligornericAβ1-42 also are characteristics of heteromeric α7β2-nAChRs, but not of homomeric α7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Aβ when elicited from MS/DB neurons derived from nAChR β2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric α7β2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Aβ1-42 supports the existence of mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and could be targeted by disease therapies.
Neuron dissociation and patch clamp recordings were performed as described in (Wu et al., 2002; Wu et al., 2004b). Briefly, each postnatal 2-4 week-old Wistar rat or mouse (wild-type C57/B16 or nAChR β2 knockout mice on a C57/B16 background kindly provided by Dr. Marina Picciotto, Yale University) was anesthetized using isoflurane, and the brain was rapidly removed. Several 400-μm coronal slices, which contained the medial septum/diagonal band (MS/DB) or the ventral tegmental area (VTA), were cut using a vibratome (Vibratorne 1000 plus; Jed Pella Inc., Redding, Calif.) in cold (2-4° C.) artificial cerebrospinal fluid (ACSF) and continuously bubbled with carbogen (95% O2-5% CO2). The slices were then incubated in a pre-incubation chamber (Warner Ins., Holliston, Mass.) and allowed to recover for at least 1 h at room temperature (22±PC) in oxygenated ACSF. Thereafter, the slices were treated with pronase (1 mg/6 mL) at 31° C. for 30 min and subsequently treated with the same concentration of thermolysin for another 30 min. The MS/DB or VTA region was micropunched out from the slices using a well-polished needle. Each punched piece was then dissociated mechanically using several fire-polished micro-Pasteur pipettes in a 35-mm culture dish filled with well-oxygenated, standard external solution (in mM: 150NaCl, 5KCl, 1MgCl2, 2CaCl2, 10 glucose 10, and 10 HEPES; pH 7.4 (with Tris-base). The separated single cells usually adhered to the bottom of the dish within 30 min. Perforated-patch whole-cell recordings coupled with a U-tube or two-barrel drug application system were employed (Wu et al., 2002). Perforated-patch recordings closely maintain both intracellular divalent cation and cytosolic element composition (Horn and Marty, 1988). In particular, perforated-patch recording was used to maintain the intracellular ATP concentration at a physiological level. To prepare for perforated-patch whole-cell recording, glass microelectrodes (GC-1.5; Narishige, East Meadow, N.Y.) were fashioned on a two-stage vertical pipette puller (P-830; Narishige, East Meadow, N.Y.), and the resistance of the electrode was 3 to 5 MΩ when filled with the internal solution. A tight seal (>2 GΩ) was formed between the electrode tip and the cell surface, which was followed by a transition from on-cell to whole-cell recording mode due to the partitioning of amphotericin B into the membrane underlying the patch. After whole-cell formation, an access resistance lower than 60 MΩ was acceptable during perforated-patch recordings in current-clamp mode, and an access resistance lower than 30 MΩ was acceptable during voltage-clamp recordings. The series resistance was not compensated in the experiments using dissociated neurons. Under current-clamp configuration, membrane potentials were measured using a patch-clamp amplifier (200B; Axon Instruments, Foster City, Calif.). Data was filtered at 2 kHz, acquired at 11 kHz, and digitized on-line (Digidata 1322 series A/D board; Axon Instruments, Foster City, Calif.). All experiments were performed at room temperature (22±1° C.). The drugs used in the present study were GABA, glutamate, ACh, choline, methyllycaconitine (MLA), dihydro-β-erythroidine (DHβE), muscarine (all purchased from Sigma-Aldrich, St. Louis, Mo.), RJR-2403 (purchased from Tocris Cookson Inc., Ballwin, Mo.), and Aβ1-42 and scrambled Aβ1-42 (purchased from rPeptide, Athens, Ga.).
Riboprobe construction: Templates for in vitro transcription were created using PCR and sense or antisense primers spanning the 5′ SP6 promoter or the 3′ T7 promotor, respectively (α7 subunit: 5′-atttaggtgacaetatagaagnggatcatcgtgggcctetcagtg-3′ (SEQ. 1D. NO.: 1) and 5′-taatacgactcactatagggagagaggcgatgtageggacctc-3′ (SEQ. ID. NO.: 2); β2 subunit: 5′-atttaggtgacactatagaagngtcacggtgttectgctgctcatct-3′(SEQ. ID. NO.: 3) and 5′-taatacgactcactatagggagatcctccetcacactctggtcatca-3′ (SEQ. ID. NO.: 4)). Antisense or sense probes were then created by in vitro transcription using SP6 or T7 polymerases, respectively, and by incorporation of biotin-tagged UTP (for β2 subunit probes) or digoxigenin-tagged UTP (for α7 subunit probes; biotin or digoxigenin RNA labeling mix; Roche Applied Science, Indianapolis, Ind.). 433 bp or 520 bp products corresponded to mRNA nucleotides 953-1385 for α7 subunits or mRNA nucleotides 1006-1525 for β2 subunits thus produced are highly specific to the individual subunits.
Tissue RT-PCR: RT-PCR assays followed by Southern hybridization with nested oligonucleotides were done as previously described to identify nAChR subunit transcripts and to quantify levels of expression normalized both to housekeeping gene expression and levels of expression in whole brain (Zhao et al., 2003; Wu et al., 2004), but using primers designed to detect rat nAChR subunits. The Southern hybridization technique coupled with quantitation using electronic isotope counting (Instant Imager, Canaberra Instruments, Meridien, Conn.) yielded results equivalent to those obtained using real-time PCR analysis.
Single-cell RT-PCR: Precautions were taken to ensure a ribonuclease-free environment and to avoid PCR product contamination during patch-clamp recording and single-cell collection prior to execution of RT-PCR. Single-cell RT-PCR was performed using the Superscript III CellDirect RT-PCR system (Invitrogen, Carlsbad, Calif.). Briefly, after whole-cell patch-clamp recording, single-cell content was harvested by suction into the pipette solution (˜3 μL) and immediately transferred to an autoclaved 0.2 mL PCR tube containing 10 μ of cell resuspension buffer and 1 μL of lysis enhancer. Single cells were lysed by heating at 75° C. for 10 min. Potential contaminating genomic DNA was removed by DNase I digestion at 25° C. for 6 min. After heat-inactivation of DNaseI at 70° C. for 6 min in the presence of EDTA, reverse transcription (RT) was performed by adding reaction mix with oligo(dT)2O and random hexamers and SuperSciptIII enzyme mix and then incubating at 25° C. for 10 min and 50° C. for 50 min. The reaction was terminated by heating the sample to 85° C. for 5 min. The PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and nAChR α3, α4, α7, β2 and β4 subunits were designed using the Primer 3 internet server (MIT) and assuming an annealing temperature of ˜60° C. [nearest neighbor]. PCR was performed with 20 μ
of hot-start Platinum PCR Supermix (Invitrogen, Carlsbad, Calif.), 3 μ
of cDNA template from the RT step, and 1 μ
of gene specific primer pairs (5 pmole each) with the following thermocycling parameters: 95° C. for 2 mM; (95° C. for 30 s, 60° C. for 30 s, and 72° C. for 40 s)×70 cycles, 72° C. for 1 min. PCR products were resolved on 1.5% TBE-agarose gels, and stained gels were used to visualize bands, employing digital photography and a gel documentation system to capture images.
Tissues were Dounce homogenized (10 strokes) in ice-cold lysis buffer (1% (v/v) Triton X-100, 150 mM EDTA, 10% (v/v) glycerol, 50 mM Tris-HCl, pH 8.0) containing 1× general protease inhibitor cocktails (Sigma-Aldrich, St. Louis, Mo.). The lysates were transferred to microcentrifuge tubes and further solubilized for 30 min at 4° C. The detergent extracts (supernatants) were collected by centrifugation at 15,000 g for 15 min at 4° C., and protein concentration was determined for sample aliquots using bicinchoninic acid (BCA) protein assay reagents (Pierce Chemical Co., Rockford, Ill.). The detergent extracts were then precleared with 50 μL of mixed slurry of protein A-Sepharose and protein G-Sepharose (1:1) (Amersham Biosciences, Piscataway, N.J.) twice, each for 30 min at 4° C. For each immunoprecipitation, detergent extracts (1 mg) were mixed with 1 μg of rabbit anti-α7 antisera (H302) or rabbit IgG (as immunological control) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) and incubated at 4° C. overnight with continuous agitation. Protein A-Sepharose and protein G-Sepharose mixtures (50 μL) were added and incubated at 4° C. for 1 h. The beads were washed four times with ice-cold lysis buffer containing protease inhibitors. Laemmli sample buffer eluates were resolved by SDS-PAGE. Proteins were transferred onto Hybond ECL nitrocellular membranes (Amershan Biosciences, Sunnyvale, Calif.). The membranes were blocked with TBST buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (v/v) Tween 20) containing 2% (w/v) non-fat dry milk for at least 2 h and incubated with rat monoclonal anti-β2 antibody (mAb270; Santa Cruz, Calif.) or anti-α7 antisera (H302), respectively, at 4° C. overnight. After three washes in TBST, the membranes were incubated with goat anti-rat or goat anti-rabbit secondary antibodies (1:10,000) (Pierce Chemical Co., Rockford, Ill.) for 1 h and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, Ill.).
cDNAs encoding rat α7 and β2 subunits were amplified by PCR with pfuUltra DNA polymerase and subcloned into an oocyte expression vector, pGEMHE, with T7 orientation and confirmed by automated sequencing. cRNAs were synthesized by standard in vitro transcription with T7 RNA polymerase, confirmed by electrophoresis for their integrity, and quantified based on optical absorbance measurements using an Eppendorf Biophotometer.
Oocyte preparation and cRNA injection: Female Xenopus laevis (Xenopus I, Ann Arbor, Mich.) were anesthetized using 0.2% MS-222. The ovarian lobes were surgically removed from the frogs and placed in an incubation solution consisting of (in mM): 82.5NaCl, 2.5KCl, 1MgCl2, 1CaCl2, 1Na2HPO4, 0.6 theophylline, 2.5 sodium pyruvate, 5 HEPES, 50 mg/mL gentamycin, 50 U/mL penicillin and 50 μg/mL streptomycin; pH 7.5. The frogs were then allowed to recover from surgery before being returned to the incubation tank. The lobes were cut into small pieces and digested with 0.08 Wunsch U/mL liberase blendzyme 3 (Roche Applied Science, Indianapolis, Ind.) with constant stirring at room temperature for 1.5-2 h. The dispersed oocytes were thoroughly rinsed with incubation solution. Stage VI oocytes were selected and incubated at 16° C. before injection. Micropipettes used for injection were pulled from borosilicate glass (Drummond Scientific, Broomall, Pa.). cRNAs encoding α7 or β2 at proper dilution were injected into oocytes separately or in different ratios using a Nanoject microinjection system (Drummond Scientific, Broomall, Pa.) at a total volume of ˜20-60 nL.
Two-electrode voltage-clamp recording: One to three days after injection, an oocyte was placed in a small-volume chamber and continuously perfused with oocyte Ringer's solution (OR2), consisting of (in mM): 92.5NaCl, 2.5KCl, 1CaCl2, 1MgCl7 and 5 HEPES; pH 7.5. The chamber was grounded through an agarose bridge. The oocytes were voltage-clamped at −70 mV to measure ACh (or choline)-induced currents using GeneClamp 500B (Axon Instruments, Foster City, Calif.).
Dissociated MS/DB neurons were fixed with 4% paraformaldehyde for 5 min, rinsed three times with PBS, and treated with saponin (1 mg/mL) for 5 min as a permeabilizing agent. After rinsing four times with PBS, the neurons were incubated at room temperature in anti-choline acetyltransferase (ChAT) primary antibody (AB305; Chemicon International, Temecula, Calif.) diluted 1:400 in Hank's balanced salt solution (supplemented with 5% bovine serum albumin as a blocking agent) for 30 min. Following another three rinses with PBS, a secondary antibody (anti-mouse IgG; Sigma-Aldrich) was applied at room temperature for 30 min (diluted 1:100). After rinsing a final three times with PBS, the labeled cells were visualized using a Zeiss fluorescence microscope (Zeiss, Oberkochen, Germany), and images were processed using Photoshop (Adobe Systems Inc., San Jose, Calif.). For double immunolabeling of α7 and β2 subunits of nAChRs on single dissociated MS/DB neurons, the following antibodies were used: a rabbit antibody (AS-5631S, 1:400; R and D, Las Vegas, Nev.) against α7 subunit, a rat antibody against β2 subunit (Ab24698, 1:500; Abeam, Cambridge, Mass.), Alexa Fluor 594-conjugated anti-rabbit IgG, and Alexa Fluor 488-conjugated anti-rat IgG; (1:300; Molecular Probes, Calif.).
Aβ preparation: Amyloid β peptides (Aβ1-42) were purchased from rPeptide Corn (Athens, Ga.). As previously described (Wu et al., 2004a), some preparations involved reconstitution of Aβ peptides per vendor specifications in distilled water to a concentration of 100 μM, stored at −20° C., and used within 10 days of reconstitution. These thawed peptide stock solutions were used to create working dilutions (1-100 nM) in standard external solution before patch-clamp recording. Working dilutions were used within 4 hours before being discarded. Atomic force microscopy (AFM) was employed to define and analyze over time the morphology of prepared Aβ1-42. Aliquots of freshly prepared samples of Aβ1-42 diluted in standard external solution were spotted on freshly cleaved mica. After 2 min the mica was washed with 200 μL of deionized water, dried with compressed nitrogen, and completely air-dried under vacuum. Images were acquired in air using a multimode AFM nanoscope IIIA system (Veeco/Digital Instruments, Plainview, N.Y.) operating in the tapping mode using silicon probes (Olympus, Center Valley, Pa.).
Protocols to obtain different forms of Aβ1-42: Different conditions were utilized to specifically prepare monomeric, oligomeric or fibrillar forms of Aβ1-42.
Monomers: Aβ1-42 was reconstituted in DMSO to a concentration of 100 μM and stored at −80° C. For each use, an aliquot of stock sample was freshly thawed and diluted into standard extracellular solution as above just before patch recordings and used for no more than 4 h. This protocol yielded a predominant, monomeric form.
Oligomers: Aβ1-42 reconstituted in distilled water to a concentration of 100 μM and stored at −80° C. was used within 7 d of reconstitution. Aliquots diluted in standard extracellular solution and used within 4 h yielded a predominantly oligomeric form.
Fibrils: Aliquots of Aβ1-42 stock solution (water dissolved to 100 μM) were thawed and incubated at 37° C. for 48 h at low pH (pH=6.0). Working stocks diluted in standard extracellular solution yielded a predominantly fibrillar form.
Genomic DNA from mice newly born to heterozygotic, nAChR β2 subunit knockout parents was extracted from mouse tail tips using the QIAgen DNeasy Blood & Tissue Kit following the manufacture's protocol. PCR amplification of the nAChR β2 subunit or lac-Z (an indicator for the knockout) were performed using the purified genomic DNA as template and gene specific primer pairs (forward primer: CGG AGC ATT TGA ACT CTG AGC AGT GGG GTC GC (SEQ. ID. NO.: 5); backward primer: CTC GCT GAC ACA AGG GCT GCG GAC (SEQ. ID. NO.: 6); lac-Z forward primer: CAC TAC GTC TGA ACG TCG AAA ACC CG (SEQ. ID. NO.: 7); backward primer: CGG GCA AAT AAT ATC GGT GGC CGT GG (SEQ. ID. NO.: 8)) with annealing at 55° C. for 1 min and extension at 72° C. for 1 min for 30 cycles with GO Taq DNA polymerase (Promega, Madison, Wis.). PCR products were resolved on 1% agarose gels and stained for visualization before images were captured using digital photography.
An initial series of experiments identified cholinergic neurons acutely dissociated from rat MS/DB (
The inventors next tested for the presence of functional nAChRs on MS/DB cholinergic neurons. Under voltage-clamp recording conditions, rapid application of 1 mM ACh induced inward current responses with relatively rapid activation and desensitization kinetics (
With regard to relatively slow kinetics of α7-nAChR-like responses in MS/DB cholinergic neurons due to co-assembly of α7 with other nAChR subunits, the inventors performed relative quantitative RT-PCR analysis of nAChR subunit expression as messenger RNA in MS/DB compared to whole-brain and VTA tissues. The results demonstrated that nAChR α7 and β2 subunits were among those co-expressed regionally (
Pharmacological approaches were used to compare features of functional nAChRs in MS/DB cholinergic or VTA DAergic neurons. The α7-nAChR-selective antagonist, MLA showed similar antagonist potency toward choline-induced currents in either MS/DB (FIG. 4Aa) or VTA (FIG. 4Ab) neurons. Analysis of concentration-inhibition curves (FIG. 4Ac) yielded IC50 values and Hill coefficients of 0.7 nM and 1.1, respectively, for MS/DB neurons (n=8) and 0.4 nM and 1.2, respectively, for VTA neurons (n=9, MS/DB vs. VTA p>0.05). However, the β2*-nAChR-selective antagonist, DHβE was ˜500-fold less potent as an inhibitor of choline-induced current in MS/BD neurons (FIG. 4Ba) than in VTA neurons (
Basal forebrain cholinergic neurons are particularly sensitive to degeneration in AD. To demonstrate that novel α7β2-nAChRs on MS/DB cholinergic neurons are involved, the inventors determined the effects of Aβ1-42 on these receptors. The experimental protocol involved repeated, acute challenges with 10 mM choline, and control studies in the absence of peptide demonstrated that there was no significant rundown of such responses when spaced at a minimum of 2-min intervals (FIG. 5Aa). During a continuous exposure to 1 nM Aβ1-42 starting just after an initial choline challenge and continuing for 10 min, responses to choline challenges were progressively inhibited with time, although reversibly so as demonstrated by response recovery after 6 min of peptide washout (FIG. 5Ab). By contrast, exposure to 1 nM scrambled Aβ1-42 (as a control peptide) had no effect (FIG. 5Ac). Choline-induced currents in dissociated VTA DAergic neurons were not sensitive to 1 nM Aβ1-42 treatment (FIG. 5Ad). Quantitative analysis of several replicate experiments (
The inventors' previous studies indicated that α4β2-nAChRs were more sensitive to Aβ1-42 than homomeric α7-nAChRs (Wu et al., 2004a). Concentration dependence of effects of Aβ1-42 on choline-induced currents in MS/DB neurons was evident, with effects being negligible at 0.1 nM and effects at 1 nM being about half of those observed for 10 nM peptide (
To further investigate features of presumed, novel α7β2-nAChRs as naturally expressed in basal forebrain cholinergic neurons, the inventors introduced nAChR α7 subunits alone or in combination with β2 subunits into Xenopus oocytes. Compared to homomeric α7-nAChRs (FIG. 7Aa), heteromeric α7β2-nAChRs expressed in oocytes injected with rat nAChR α7 and β2 subunit cRNAs at a ratio of 1:1 exhibited smaller peak current responses to choline and slower current decay rates (FIG. 7Ab). These results are consistent with findings in a previous report (Khiroug et al., 2002). As was the case for comparisons between native nAChR responses in rat MS/DB or VTA neurons (
As further support for the concept that basal forebrain cholinergic neurons express novel α7β2-nAChRs, the inventors used wild-type and nAChR β2 subunit knockout (β2−/−) mice. PCR genotyping was used to identify wild-type or β2−/− mice (
nAChRs in basal forebrain participate in cholinergic transmission and cognitive processes associated with learning and memory (Levin and Rezvani, 2002; Mansvelder et al., 2006). During the early stages of AD, decreases in nAChR-like radioligand binding sites have been observed (Burghaus et al., 2000; Nordberg, 2001), suggesting that nAChR dysfunction could be involved in AD pathogenesis and cholinergic deficiencies (Nordberg, 2001). Evidence indicates that enhancement of α7-nAChR function protects neurons against A toxicity through any or some combination of a number of different mechanisms, as outlined previously (Sadot et al., 1996; Lahiri et al., 2002; Nagele et al., 2002; Geerts, 2005; Liu et al., 2007a). On the other hand, pharmacological interventions or diminished nAChR expression produces learning and memory deficits (Levin and Rezvani, 2002).
Findings described herein are consistent with the natural expression of a novel, heteromeric, functional α7β2-nAChR subtype on forebrain cholinergic neurons that is particularly sensitive to functional inhibition by a pathologically-relevant concentration (1 nM) of Aβ1-42. Some previous studies investigating the acute effects of Aβ1-42 on nAChRs examined receptors on neurons from regions other than the basal forebrain or that were heterologously expressed (Liu et al., 2001; Pettit et al., 2001; Grassi et al., 2003; Wu et al., 2004a; Lamb et al., 2005; Pym et al., 2005) and/or used Aβ peptides at concentrations (between 100 nM and 10 μM) that greatly exceed Aβ concentrations found in AD brain (Kuo et al., 2000; Mehta et al., 2000). Other studies identified α7-nAChR-like, ACh-induced currents in MS/DB cholinergic neurons using slice-patch recordings (Henderson et al., 2005; Thinschmidt et al., 2005) and characterized functional, non-α7-nAChRs using acutely-dissociated forebrain neurons (Fu and Jhamandas, 2003). Studies described herein combined whole-cell current recordings from acutely-dissociated neurons and investigation of MS/DB cholinergic neuronal nAChRs to identify functional nAChRs that have some features of receptors containing α7 subunits, but also found high sensitivity of these nAChRs to low concentrations of Aβ1-42. Studies described herein are consistent with other previous findings and also indicate that functional α7β2-nAChRs can be heterologously expressed in oocytes. Histological studies have demonstrated co-expression of nAChR α7 and β2 subunits in most forebrain cholinergic neurons (Azam et al., 2003). The results also are consistent with those observations and show cell-specific, co-expression of nAChR α7 and β2 subunits at both message and protein levels. There are other reports (Yu and Role, 1998); (El-Hajj et al., 2007) that nAChR α7 subunits could be co-assembled with other subunits to form native, heteromeric, α7*-nAChRs. These findings herein are consistent with those observations. The notion that the Aβ1-42-sensitive, functional nAChR subtype in MS/DB neurons displaying some features of nAChRs containing α7 subunits, but distinctive from homomeric α7-nAChRs, is composed of α7 and β2 subunits, is supported by the loss of Aβ sensitivity and the conversion of functional nAChR properties to those like homomeric α7-nAChRs in nAChR β2 subunit knockout animals. It has been reported that there are two isoforms (α7-1 and α7-2) of α7-nAChR transcript in homomeric α7-nAChRs. The α7-2 transcript that contains a novel exon is widely expressed in the brain and showed very slow current kinetics (Severance et al., 2004); (Severance and Cuevas, 2004); (Saragoza et al., 2003). However, the inventors contend that the heteromeric α7β2-nAChR described in the present study and expressed in MS/DB neurons is not a homomeric nAChR composed of or containing the α7-2 transcript for three reasons: (1) in β2−/− mice, α7-nAChR-like whole-cell current responses to choline acquire fast kinetic characteristics like those of α7-nAChR responses in VTA neurons, (2) immunoprecipitation-western blot analyses show co-assembly of α7 and β2 subunits from the MS/DB but not from the VTA, nor from the MS/DB of β2−/− mice, and (3) pharmacologically heteromeric α7β2-nAChRs were sensitive not only to MLA, but also to DHβE.
A recent study suggested that levels of oligomeric forms of Aβ1-42, rather than monomers or Aβ fibrils, most closely correlate with cognitive dysfunction in animal models of AD (Haass and Selkoe, 2007). The inventors' findings also convey that Aβ oligomers have the most profound effects on nAChR function, thus extending earlier studies of Aβ-nAChR interactions (Wu et al., 2004a) and illuminating why there have been apparent discrepancies in some of the earlier work concerning Aβ-nAChR interactions.
Alzheimer's disease (AD) is a dementing, neurodegenerative disorder characterized by accumulation of amyloid β (Aβ) peptide-containing neuritic plaques, degeneration of basal forebrain cholinergic neurons, and gradually impaired learning and memory (Selkoe, 1999). The extent of learning and memory deficits in AD is proportional to the degree of forebrain cholinergic neuronal degeneration, and the extent of Aβ deposition is used to characterize disease severity (Selkoe, 1999). Processes such as impaititient of neurotrophic support and disorders in glucose metabolism have been implicated in cholinergic neuronal loss and AD (Dolezal and Kasparova, 2003). However, clear neurotoxic effects of Aβ across a range of in vivo and in vitro models suggest that Aβ plays potentially causal roles in cholinergic neuronal degeneration and consequent learning and memory deficits (Selkoe, 1999).
Based on the findings described herein, selective, high-affinity effects of oligomeric Aβ1-42 on basal forebrain, cholinergic neuronal α7β2-nAChRs acutely contribute to disruption of cholinergic signaling and diminished learning and memory abilities (Yan and Feng, 2004). Moreover, to the extent that basal forebrain cholinergic neuronal health requires activity of α7β2-nAChRs, inhibition of α7β2-nAChR function by oligomeric Aβ1-42 can lead to losses of trophic support for those neurons and/or their targets, and cross-catalyzed spirals of receptor functional loss and neuronal degeneration also can contribute to the progression of AD. Drugs targeting α7β2-nAChRs to protect them against Aβ effects or restoration of α7β2-nAChR function in cholinergic forebrain neurons will serve as viable therapies for AD.
Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).
Accordingly, the invention is not limited except as by the appended claims.
This invention was made with government support under Contract No. ROI DA015389 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/22424 | 1/28/2010 | WO | 00 | 7/27/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61148010 | Jan 2009 | US |
