Introduction
Several methods for silencing neuronal electrical activity in behaving animals have been developed that could eventually be useful for treating neurological disorders including Parkinson disease, addiction, epilepsy, depression, and chronic pain states (1 Generally, these techniques involve overexpressing an exogenous receptor in molecularly or spatially defined populations of neurons and applying a specific stimulus to activate this receptor and thereby silence the neurons.
The two most successful strategies to date have proved to be bacterial halorhodopsin and the Drosophila allatostatin receptor. Halorhodopsin is a light-activated inward chloride pump, which, when exogenously expressed in neurons, hyperpolarizes neurons rapidly and reversibly upon the application of intense yellow light Although this has been employed successfully to correlate neuronal activity with behavior
, it is limited by 1) the requirement for strong overexpression and 2) the necessity to deliver intense light to neurons in vivo. The recent development of more efficient light-driven outward proton pumps may address the first limitation
The alternate approach employs a Drosophila G protein-coupled receptor that is activated by the insect neuropeptide allatostatin When expressed exogenously in mammalian neurons, allatostatin receptor activation activates G protein-coupled inwardly rectifying potassium channels via the direct binding of G protein βγ subunits, thereby producing hyperpolarization and cessation of spiking activity. Although this has been successfully used in a number of laboratories and has been validated in behaving mammals it is limited by the requirement to directly inject allatostatin into required brain regions.
The halorhodopsin and allatostatin approaches are not ideal for human therapy due to their use of exogenous nonhuman (even nonvertebrate) receptors and the inconvenience of applying stimuli directly to the target neurons. The second of these issues can be addressed by a third silencing approach, which employs a Caenorhabditis elegans αβ heteromeric glutamate-gated chloride channel receptor (GluClR) mutated to abolish sensitivity to glutamate while retaining low nanomolar sensitivity to ivermectin (12 . In this approach, ivermectin is injected systemically and crosses the blood-brain barrier to activate a chloride flux in neurons expressing exogenous GluClRs, thereby shunting excitatory impulses. Ivermectin is a well tolerated anti-helminthic that is approved by the FDA for a variety of human parasitic infections ( )However, there are three main limitations with the ivermectin-GluClR method. First, GluClR silencing is slowly reversible, requiring days, as opposed to minutes for allatostatin and milliseconds for light-activated proton pumps. Second, use of this method has not yet spread beyond the originating laboratory (15 ), possibly due to poor functional expression stemming from the requirement to coexpress two different subunits in target neurons. Finally, as with the other silencing methods described above, GluClR exists only in lower phyla, which does not bode well for its eventual use in human clinical practice.
EXPERIMENTAL PROCEDURES
Molecular Biology
The human α1, α2, α3, and β GlyR subunit cDNAs were each subcloned into the pcDNA3.1 plasmid vector. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and the successful incorporation of mutations was confirmed by DNA sequencing.
HEK-293 Cell Culture and Transfection
HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing Serum Supreme (Lonza, Walkersville, MD) and penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO) and split onto coverslips in 3-cm dishes. On the following day, cells were transiently transfected with the relevant GlyR cDNAs via a calcium phosphate method. These cells were also transfected with empty pEGFP vector (Clontech, Mountainview, CA, USA) as a fluorescent transfection marker. In experiments investigating α1β heteromeric GlyRs, α1 and β were transfected in a 1:4 ratio.
Neuron Dissociation, Culture, and Transfection
Experiments were performed on P0–P3 male Wistar rats in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes with approval from the Animal Ethics Committee for the University of Queensland. Animals were anesthetized with isoflurane and decapitated. Brains were immediately removed, and hippocampi were dissected out. Neurons were dissociated by incubation in 0.1% trypsin/EDTA (Invitrogen) for 7 min at 37 °C and were washed in HEPES-buffered minimum essential medium with 0.014% trypsin inhibitor (type I-S from soybean; Sigma-Aldrich). After diluting the cells in neurobasal A medium (Invitrogen) supplemented with 2% B27 (Invitrogen) and 0.5 mm l-glutamine, 2 × 105 cells were added to each well of 4-well dishes (BD Falcon). Each well contained a sterilized glass coverslip treated with poly d-lysine (Invitrogen), on which the cells grew. After 1 h, this medium (conditioned medium) was removed, and 0.5 ml of medium was added, consisting half of conditioned medium and half of neurobasal A, 2% B27, penicillin/streptomycin, 0.5 mm l-glutamine, and 10 ng/ml basic fibroblast growth factor (Invitrogen) (growth medium). After 3 days, cells were washed with phosphate-buffered saline, and 0.5 ml of medium was added, consisting of 20% conditioned medium and 80% fresh growth medium. On days P7–P10, cells were washed in phosphate-buffered saline and incubated for 1 h in fresh growth medium. Transfections were performed after 4–7 days in vitro, with recordings performed 2 days later.
Electrophysiology and Data Analysis
HEK-293 cells on coverslips were placed in a bath and visualized with an inverted fluorescence microscope. Cells expressing GlyRs were identified by green fluorescent protein fluorescence. Patch clamp pipettes were pulled from borosilicate glass tubes (Vitrex, Modulohm, Denmark) on a horizontal puller (P97, Sutter Instruments, Novato, CA) and had tip resistances of 1–2 megohms when filled with the pipette solution which consisted of: 145 mm CsCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, and 10 mm EGTA, pH 7.4. Bath solution consisted of 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, and 10 mm glucose, pH 7.4. After establishment of the whole-cell recording configuration, cells were voltage clamped at −40 mV (unless otherwise indicated), and membrane currents were recorded using an Axon Multiclamp 700B amplifier and pClamp10 software (Molecular Devices, Sunnyvale, CA). Currents were filtered at 500 Hz and digitized at 2 KHz.
Ivermectin, moxidectin, emamectin benzoate, eprinomectin, and picrotoxin were all obtained from Sigma-Aldrich. Doramectin was obtained from Toronto Research Chemicals (North York, ON, Canada). Stocks of 10 mm ivermectin or its analogues, dissolved in dimethyl sulfoxide (Sigma-Aldrich), were prepared every 2 weeks and stored at −20 °C. Picrotoxin stocks were made at 100 mm in dimethyl sulfoxide and kept at −20 °C. Solutions for experiments were prepared from these stocks on the day of recording. Solutions were applied to cells via gravity forced perfusion via parallel microtubules and manual control of this system via a micromanipulator effected solution exchange in ∼ 250 ms. Experiments were conducted at room temperature (19–22 °C).
RESULTS
To identify the most appropriate clone on which to base a silencing receptor, we compared the glycine and ivermectin sensitivities of human α1, α1β, α2, and α3 GlyRs. Sample glycine- and ivermectin-gated currents in HEK-293 cells expressing α1 GlyRs are shown in Fig. 1, A and B, respectively. Because ivermectin currents are irreversible ), we generated ivermectin dose-response relationships by successively applying higher ivermectin concentrations. Averaged glycine and ivermectin dose-response curves for α1, α1β, α2, and α3 GlyRs are shown in Fig. 1, C and D. The parameters of best curve fit for these and all other mutant GlyRs examined in this study are summarized in Table 1. As glycine and ivermectin sensitivity did not vary dramatically among the four wild type (WT) constructs tested, we employed the α1 GlyR (the standard model system for structure-function studies) as a starting point for developing an improved ivermectin-activated silencing receptor.
As there are no clues to date concerning the location of the GlyR ivermectin binding site, we generated and screened a cDNA library comprising ∼1600 randomly mutated α1 GlyR clones and searched for mutations that affected ivermectin sensitivity. Library mutation frequency was selected so that each clone contained an average of 1–2 amino acid mutations. The methods for generating this library and screening it in HEK-293 cells for ivermectin sensitivity have recently been described in detail (
The screen identified one mutation, T258S, that significantly increased GlyR ivermectin sensitivity. This residue is well known to contribute to the lining of the channel pore and to form the pore binding site for several GlyR-active pharmacological modulators . Electrophysiological analysis in HEK-293 cells confirmed that the T258S mutation significantly increased receptor sensitivity to both glycine (Fig. 2, A and E) (Table 1) and ivermectin (Fig. 2, B and F and Table 1). Indeed, the T258S mutation reduced the ivermectin EC50 from ∼1.7 μm to 90 nm. As the T258S mutant GlyR was activated significantly by 10 nm ivermectin (Fig. 2, B and F), we considered it a potential candidate silencing receptor. We therefore introduced the F207A glycine binding site mutation to eliminate sensitivity to the endogenous agonist glycine. As shown in Fig. 2C, a 10 mm concentration of glycine activated no detectable current in the F207A mutant GlyR, although robust ivermectin-gated currents were observed (Fig. 2C). Unfortunately, this mutant receptor showed a significant decrease in ivermectin sensitivity relative to the WT α1 GlyR (Fig. 2F and Table 1). Thus, it is not surprising that the double mutant T258S/F207A GlyR also exhibited a significantly reduced ivermectin sensitivity relative to the T258S GlyR (Fig. 2, D and F, and Table 1). Indeed, as the ivermectin sensitivity of the double mutant was not significantly different to WT, this construct was discarded as a candidate silencing receptor. Nevertheless, these results imply a long range allosteric coupling between T258S and the ivermectin binding site similar to that previously shown between the residue corresponding to L259S and the acetylcholine binding site of the nicotinic acetylcholine receptor
The random mutant library screen also identified several mutants that decreased or eliminated ivermectin sensitivity. One of these mutations was A288T in the third transmembrane domain. Electrophysiological analysis in HEK-293 cells confirmed that the A288T mutant GlyR retained WT-like sensitivity to glycine but was almost completely insensitive to ivermectin (Fig. 3A and Table 1). As different mutations to Ala288 have contrasting effects on GlyR sensitivity to ethanol we investigated the effects of several different Ala288 mutations on ivermectin sensitivity. Whereas most tested substitutions abolished ivermectin sensitivity (data not shown), the A288G substitution resulted in a dramatic increase in sensitivity to both glycine and ivermectin (Fig. 3, C and D and Table 1).
DISCUSSION
The silencing receptor developed here exhibits a similar ivermectin sensitivity to the C. elegans αβ heteromeric GluClR (13. Because that receptor is activated by concentrations of ivermectin that reach the brain following systemic injection , we expect the F207A/A288G α1 GlyR to respond similarly. However, our human GlyR-based receptor has three main advantages over the GluClR-based silencing receptor. First, it has a larger single channel conductance implying a high inhibitory conductance per expressed receptor. Second, it expresses as a homomer, obviating the requirement to recombinantly express two different cDNAs in each target neuron. Both these measures are likely to result in an improved silencing efficiency in vivo. Finally and perhaps most importantly, the silencing construct described here is derived from a human receptor, making it a promising candidate for clinical applications. Furthermore, as the A288T mutant α1 GlyR exhibits normal WT glycine sensitivity but is insensitive to ivermectin, it should be useful as a control for exogenous receptor expression. With the halorhodopsin, allatostatin, and ivermectin-GluClR approaches, it is currently not possible to control for the expression per se of the silencing construct.
It is conceivable that other ivermectin analogues may be preferable as silencing receptor ligands in terms of their side effect profiles, pharmacokinetics, ability to cross the blood-brain barrier, and silencing receptor potency. Of the analogues investigated here, only moxidectin exhibited a higher potency than ivermectin, although the difference was not statistically significant (Table 2). Moxidectin is a milbemycin, which are aglycones of the avermectins, and these have remarkably fewer side effects than avermectins such as ivermectin . Indeed, it is predominantly for this reason that moxidectin is currently being tested in human clinical trials for river blindness . Relative to ivermectin, moxidectin exhibits a longer retention time in vivo which is a disadvantage in research experiments where rapid recovery is preferred, but it may be an advantage in many clinical settings. To date, it appears that there has been no comparative study of the ability of moxidectin and ivermectin to cross the blood-brain barrier. However, ivermectin is a potent substrate of P-glycoprotein, which efficiently pumps it out of the brain. Because moxidectin is a poor substrate for P-glycoprotein it is feasible that it may reach higher concentrations in the brain. In this respect, it is of interest to note that moxidectin crosses the blood-brain barrier in two strains of senescence-accelerated mice with particularly high efficiency, although the mechanism remains unknown Given all these considerations, moxidectin may be worthy of consideration as an improved in vivo agonist of the F207A/A288G mutant α1 Gly
Credited to World wide data science
留言