USING XENOPUS OOCYTES IN NEUROLOGICAL DISEASE DRUG DISCOVERY

Abstract

Introduction:

Neurological diseases present a difficult challenge in drug discovery. Many of the current treatments have limited efficiency or result in a variety of debilitating side effects. The search of new therapies is of a paramount importance, since the number of patients that require a better treatment is growing rapidly. As an in vitro model, Xenopus oocytes provide the drug developer with many distinct advantages, including size, durability, and efficiency in exogenous protein expression. However, there is an increasing need to refine the recent breakthroughs.

Areas covered:

This review covers the usage and recent advancements of Xenopus oocytes for drug discovery in neurological diseases from expression and functional measurement techniques to current applications in Alzheimer’s disease, painful neuropathies, and amyotrophic lateral sclerosis (ALS). The existing limitations of Xenopus oocytes in drug discovery are also discussed.

Expert opinion:

With the rise of aging population and neurological disorders, Xenopus oocytes, will continue to play an important role in understanding the mechanism of the disease, identification and validation of novel molecular targets, and drug screening, providing high-quality data despite the technical limitations. With further advances in oocytes-related techniques toward an accurate modeling of the disease, the diagnostics and treatment of neuropathologies will be becoming increasing personalized.

1. INTRODUCTION

Neurological and neuropathic diseases present as a multitude of symptoms such as dementia, stroke, and loss of motor function, chronic pain or alterations in sensation. Despite the strong research effort toward the development of treatments, large and growing populations suffer from more than 600 diseases of the nervous system including Parkinson’s disease, Alzheimer’s disease, epilepsy, schizophrenia, peripheral neuropathies, or multiple sclerosis. In the US alone, these conditions affect over 10 million people and account for almost $800 billion dollars in annual costs [1]. The large prevalence of neuropathologies and difficulties in their treatments is partly due to the gaps in the understanding of the basic cellular and biochemical mechanisms involved, which hamper the development of more effective treatments [2-4]. Moreover, brain, spine and nerve disorders often involve multiple, related dysfunctions such as changes in cellular structure, gain or loss of function mutations, or ion imbalances [5]. Understanding the interplay of these changes that ultimately lead to the disease presents a challenge for the drug developers that are looking for a distinct target to prevent or treat the disease.

Ion channels act as entryways for ions to penetrate the otherwise impermeable cell membrane. Along with supporting regulatory and other proteins, ion channels establish a connection between the environment and the neurons. The opening and closing of these channels are highly regulated. Ion channels respond only to specific signals, such as binding of a neurotransmitter, alteration in electrical membrane potential, phosphorylation of one or more subunits, and so on. Malfunctioning ion channels are the major common causes of a large number of neurological disorders [6, 7]. Aberrant K⁺ and other ion channels are responsible for neuromyotonia [8], episodic ataxia type-1 [9] and benign familial neonatal convulsions [10] among many other genetic and acquired neurological disorders [11]. Defects in voltage-gated Na⁺ channels lead to pain [12] and epilepsy [13]. Calcium channels are accountable for several psychiatric disorders [14]. Advancement in the study of ion channels has made it possible to evaluate channel dysfunctions at the various levels from the single channel to the complex neuronal systems, and the whole organism.

Ion channel activity has also been known to be regulated by pharmaceutical drugs. Given the importance of these channels in coordinating and controlling neural activity, the macromolecules forming the channels, along with some related regulatory proteins, represent key drug targets for the broad spectrum of neurological and psychiatric diseases. However, testing the specificity of drugs to regulate ion channels using typical mammalian neurons is complicated by the presence of a variety of endogenous channels. An established approach to address this problem is to simplify a model system by expressing individual classes of ion channels in cultured, non-neuronal cell lines, such as HEK 293 cells [15] and oocytes. Although such approaches are often criticized for being less physiological, this methodology became attractive for pharmacological testing because the non-neuronal cells are generally easier to transfect and can be studied using validated electrophysiology approaches.

Because of the absence of endogenous ion channels and related receptors and enzymes, HEK 293 cells and Xenopus oocytes provide a low level background ‘noise’ model for studying the biology and physiology of these proteins. In addition, the simplified cell models are amendable to high-throughput techniques to accelerate the drug discovery process [16]. For example, injection of GABA receptor mRNA from chick optic lobes into Xenopus oocytes yielded functional GABA receptors that were activatable by application of GABA. These GABA receptors activated a chloride channels in the oocyte [17].

Xenopus oocytes, derived from the South African clawed frogs Xenopus laevis, are broadly used as a model for studying ion channels. Compared to the HEK 293 cells, Xenopus oocytes are a relatively recent addition to the neurologic drug development scene. When compared to other recently introduced organisms such as Caenorhabditis elegans and Danio rerio (zebrafish), Xenopus oocytes provide a few experimental advantages. Evolutionarily, Xenopus species share a closer common ancestor with mammals, sharing orthologs for 79% of identified human disease genes [18]. Additionally, Xenopus species offer many practical benefits as a single frog can produce thousands of oocytes that only require basic salt solutions to be cultured [19]. Moreover, the oocytes are physically robust and can withstand a variety of treatments, including microinjection and surgical manipulation. These attributes in addition to close to a 100% success rate in transfection, absence of endogenous ion channels, and ease in electrophysiological measurements down to single ion channels made the oocytes an attractive and reliable model for the drug discovery [20] and a useful bridge to mammalian studies.

Early investigations on Xenopus oocytes concentrated on morphological and cytological processes that occur during the growth, maturation, fertilization and the development of the embryo. A crucial step in using oocytes for future drug discovery was the demonstration by Gurdon and colleagues that showed the ability of the oocytes to synthesize exogenous proteins when injected with exogenous messenger RNA [21]. Responses to neurotransmitters were demonstrated in Xenopus oocytes in 1977 [22], and in the following years oocytes presented themselves a useful model for the study of molecular mechanisms related to neurological tasks. Most of the studies using Xenopus oocytes are centered on the ion channels expressed on the oocytes’ cell membrane and measurements of the small ionic currents that inform the function of the ion channel. Since the inception of the patch clamp technique during the early 1980s, oocytes became the mainstream model for the neurological drug discovery process.

There was, however, a general reluctance to use oocytes as representative systems for drug discovery because of their non-mammalian status and potential translational issues [23], although today most of the studies rely on using transfection materials (i.e. mRNA, cDNA) derived from humans. These translational issues stem from concerns that genes for non-endogenous proteins will be translated by the oocyte cell machinery, instead of the mammalian environment. Drugs often show different IC50 values between oocytes and mammalian cells. For example, certain anti-psychotics drugs had similar IC50 values toward stably expressed hERG potassium channels in different mammalian cells. However, these values were five to one hundred times larger when hERG was expressed transiently in Xenopus oocytes [24].

Additional hurdles in their acceptance included developing techniques for mRNA or cDNA injection into each oocyte to induce specific ion channels. The 96 well plate or other high throughput in vitro transcription assays are convenient for many mammalian cells. However, unlike with most cells, standard in vitro transcription assays are not viable with Xenopus oocytes. Instead, microinjection is traditionally used to introduce the cDNA or other genetic material into either the nucleus or cytoplasm.

Despite these translational and technical issues, the unique features of Xenopus oocytes, such as a millimeter scale size, expression compatibility, and physical robustness [25, 26] led to the development of many drugs. For example, many AMPA antagonists for the treatment of patients with epilepsy and partial-onset seizures have been developed using Xenopus oocytes [27]. Table 1 shows new investigational drugs for the treatment of neurological diseases that originated from oocytes studies and advanced to the clinical trials. Despite the growing competition from mammalian cell lines and new emerging animal models (i.e. zebrafish, C. elegans), the oocytes continue to be used as an important model of choice for the study of neurotransmitters and ion channels by many research institutions and pharmaceutical companies.

Table 1.

Neurological disease drugs undergoing clinical trials that employed Xenopus oocytes

Drug	Disease	Target	Reference
Encenicline	Alzheimer’s Disease	Nicotinic acetylcholine alpha g7 receptor (α7 nAChR)	[28]
Ethosuximide	Chemotherapy-Induced Peripheral Neuropathy (CIPN)	G protein-activated inwardly rectifying K+ channel (GIRK)	[29]
Memantine	CIPN	N-methyl-D-aspartate receptor (NMDA)	[30]
Pregabalin	Diabetic Peripheral Neuropathy	Neuronal Glutamate Transporter Type 3 (EAAT3)	[31]
Amiloride	Multiple Sclerosis	Acid-sensing ion channel (ASIC)	[32]
Padsevonil	Epilepsy	Type A gamma-aminobutyric acid receptor (GABAA)	[33]
Lacosamide	Epilepsy	Type A gamma-aminobutyric acid receptor (GABAA)	[34]

Oocytes have shown broad potential to study pathologies that involves malfunctioning ion channels and other proteins. Among the neurological disorders, intensive studies included epilepsy [35, 36], brain tumors [37], autism [38, 39], multiple sclerosis [40] and many other conditions.

This account will cover the role and application of Xenopus oocytes in the discovery of drugs that treat neuropathology. A particular emphasis will be given first to the methods of the oocytes’ transfections, allowing expression of diverse targets derived from human tissues. Another emphasis will be put on the usage of electrophysiological techniques as the major method for screening and selecting drugs. Finally, we will illustrate the utility of oocytes in drug development of several neurological applications such as Alzheimer’s disease, amyotrophic lateral sclerosis, and chronic pain, three devastating progressive neurodegenerative diseases with different origins where oocytes have a great, but somewhat not fully unrealized potential.

2. OOCYTES ARE AN EXPRESSION POWER HOUSE

2.1. Xenopus Oocytes as cell models

An oocyte is a female germ cell in the process of development. Compared to other cells, Xenopus oocytes have striking physical characteristics with visually polarized dark and light hemispheres. This separation goes deeper than just the plasma membrane as intracellular components such as the germinal vesicle, distribution of yolk platelets, maternal mRNA, membrane receptors, and any endogenous ion channels have polarized localizations [41]. This uneven distribution is typically considered when performing microinjection or membrane transporter detection. Xenopus oocytes are also incredibly large cells with an average diameter of 1.3 mm [42]. In terms of sheer volume, they greatly exceed other cells used for drug discovery. In addition to providing greater source material for biochemical assays, this size advantage gives researchers ease of access and convenience when performing delicate procedures such as microinjection or voltage clamping. This includes using two recording electrodes during two-electrode voltage clamp (TEVC). Xenopus oocytes can even accommodate a third electrode, which can be used for intracellular injection during recordings or to collect additional data [43].

In electrophysiological experiments, another important biological factor to consider in a model is interference from endogenous ion channels. As mentioned in the Introduction, the absence of endogenous ion channels is preferred to provide a “zero background” model (see “Limitations of oocytes section” describing some endogenous currents in oocytes), and Xenopus oocytes are largely free of interfering channels. Xenopus oocytes are often deposited in unfavorable environments such as streams prior to fertilization. While many cells require strict physiological conditions for survival, these oocytes do not rely on their surroundings for nutrient uptake and lack a wide variety of cell membrane transport elements. Biologically, this independence from their environment translates into Xenopus oocytes coming pre-equipped with all the necessary substrates and machinery for growth and protein expression as well as very few membrane transport elements [44]. While Xenopus still contain some endogenous channels, they contain fewer than other commonly used cells such as HEK or CHO cells, which leads to lower levels of interference with exogenously expressed proteins [45]. The convenience that comes with oocytes is preferred in many studies, where using other expression systems would require engineering knockout cell lines [46]. For this reason, as well as their size, Xenopus oocytes have been widely considered as an optimal option as an exogenous expression system.

2.2. Expression vehicle for mammalian and human proteins

Xenopus oocytes’ translational regulation and ability to confer a range of post-translation modifications are parts of what allow them to achieve native activity when expressing complex, multi-unit exogenous proteins. During oocyte development, germ cells grow rapidly and stockpile large amounts of cellular machinery for translation, including mRNA, ribosomes, tRNA, and other components. Despite these large stores, in the advanced stages of oocyte development, known as developmental stages V and VI [47], oocytes only synthesize proteins from as little as 5% of stored mRNA, and the rest is masked as non-translating mRNA [48]. Such low activity in producing endogenous proteins, makes the late stages of Xenopus oocytes an attractive model for translation as they provide all the necessary machinery for exogenous protein expression and allow exogenous mRNA to translate with little competition from endogenous elements. On top of that, there is, apparently, no preferential selection for translation of endogenous mRNA over foreign genetic material [49], including genes from other species. Outside of providing ample equipment and opportunity for exogenous protein translation, Xenopus oocytes also offer rich advantages in terms of post-translational modifications and processing. Processes like peptide cleavage, phosphorylation, glycosylation, segregation, secretion, and others are all able to be properly performed in Xenopus oocytes [50]. As a result, oocytes have been frequently utilized to express not only various channels but also their modifications and related regulatory receptors.

2.3. Expression techniques: mRNA, cDNA and virus

Since the major usage of Xenopus oocytes revolves around their ability to efficiently and reliably express exogenous proteins from mammals including humans, a substantial effort has been placed in the development of the transfection techniques. One of the common and popular transfection methods involves the microinjection of RNA directly into the cytoplasm [51].The sources of RNA can range from mRNA collected from tissues to synthetic RNA produced from a cDNA template. A schematic approach is shown in Figure 1 (from [52])

Figure 1:

Diagram of the procedures used to transplant neurotransmitter receptors from the human brain to the oocyte plasma membrane by injecting either brain cell membranes (Upper) or brain mRNA (Lower) into Xenopus oocytes. With permission from [52]. Copyright (2002) National Academy of Sciences, U.S.A

Additionally, cDNA itself can also be used for gene expression in Xenopus oocytes. This technique, however, typically involves direct microinjection into the nucleus. Even though the nucleus is large and always resides in the animal pole of the oocyte, it can still be a challenge to consistently locate the nucleus since the oocyte plasma membrane is not transparent. Techniques have been developed to make nuclear injection more reliable, such as briefly centrifuging the oocyte to reposition the nucleus [53]. Additionally, several commercially available systems provide high speed automated intracellular and intranuclear injections [54]. These systems can ease the time investment of research centers and circumvent the burden of manually injecting mass quantities of oocytes for high throughput drug screening.

There were early attempts at the automation of injection of Xenopus oocytes [55]. However, the success rate for transfection of these automated injection systems was less than 30% of the manual injection systems. These newer automated injection systems (Roboocyte [55], XenoFactor [56], and similar products) provide great utility, but they have not become commonplace in electrophysiological studies. In addition to being a new advancement, there is a significant initial cost associated with automatic injection. Beyond that, these systems are targeted for high to medium-throughput analysis, which is often beyond the scope of many studies. Today, automated electrophysiology systems for oocytes are limited to three systems (OpusExpress (Molecular Devices), Robocyte 2 and HiClamp (both from Multi Channel Systems MCS GmbH) with only Robocyte 2 and HiClamp being offered. While several publications describe the advantages of the automatic systems [57, 58], little is known about their disadvantageous despite the high initial cost of investment [59]. Among disadvantageous, we envision a higher level of false negative results due to lack of the control for individual recording as well as potential issues with the automated changes in bath solutions degrading the seals of the patched cells.

One of the early techniques that showed promise to circumvent the difficult nuclear injection associated with cDNA was a virus based [60]. The method involves an infection step with a virus expressing the T7 RNA polymerase in addition to a co-injection of cDNA into the cytoplasm. It combines the ease of obtaining cDNA with the more manageable cytoplasmic injection. However, it has not been perfected and can result in inexplicable lower levels of expression. Further work is still needed to refine an expression method that is clearly superior to either classical methods of RNA injection or cDNA nuclear injection.

2.4. Transplantation approach toward expression

The ultimate goal of using mRNA (or other isolated genetic materials) to express exogenous proteins in oocytes is to study ion channels in an environment that is as close as possible to a native organism. While Xenopus oocytes are capable of efficiently expressing these ion channels, a new, more authentic method of studying ion channels is available. Through the injection of microsomes, electrophysiological methods can be used for direct examination of the exact malfunctioning ion channels that were the source of the neurological disease. Furthermore, this method allows for a more individualized approach to studying diseases affecting ion channels.

Less common but promising methods of introducing foreign proteins into the oocyte system are based on already expressed receptors from mammalian and other cell cultures that can be directly transplanted into a Xenopus oocyte. This technique takes advantage of native mammalian expression arrangements and adds the convenience that comes with the oocyte system. In order to transplant exogenous receptors, oocytes are injected with membrane vesicles from cultured cells. After some incubation time, the transplanted receptors can be detected having been inserted by the oocyte into the plasma membrane [61]. This technique has been originally applied to express native nicotinic acetylcholine receptors after injection of ooctyes with purified Torpedo electroplaque membrane vesicles [62]. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes resulted in the successful expression of gamma-aminobutyric acid type A (GABA_A) receptors. Electrophysiological evaluation of the responses of these receptors to GABA and other mediators revealed that they were incorporated into the oocyte membrane with full retention of their original pharmacological properties, such as sensitivity to Cl^- channel blockers, benzodiazepines, and general anesthetics [63]. Recently, this approach has been applied to express synaptic proteins derived from rat brain neurolemma to study the effects of neurotoxicants on voltage-sensitive ion channels (Figure 2, from [64]).

Figure 2:

Physical incorporation of rat brain neurolemma into the plasma membrane of Xenopus oocytes: Whole eggs were injected with buffer only (Control) or with neurolemma (PND90) covalently-labeled with rhodamine and visualized under brightfield (left image) or fluorescence conditions (right image). Tissue sections of buffer-injected oocytes (Control) or injected with rhodamine-labeled neurolemma and visualized under brightfield (left image) or fluorescence conditions (right image). The plasma membrane from sectioned oocytes was double-labeled for voltage-sensitive sodium channels using a NavPAN antibody. Adapted with permission from [64].

This transplantation approach has been recently advanced to human proteins. Thus, cell membranes from the brains of humans who suffered Alzheimer’s disease have been micro-transplanted into oocytes to study the functional characteristics of ion channels and receptors from the post-mortem membrane transplants [65] (Figure 1). As micro-transplantation of ion channels into Xenopus oocytes further develops, it stands to greatly open the ability to study the effects of drug compounds on ion channels from any species in their native form.

Overall, regardless of technique, Xenopus oocytes have been found to impressively express proteins from numerous, different species with reasonable efficiency, reliability, and reproducibility thus providing excellent starting point for ion channel evaluations and drug screening.

3. FUNCTIONAL MEASUREMENT OF OOCYTES FOR DRUG DISCOVERY

3.1. Electrophysiology as a major tool for studying oocytes

Xenopus oocytes have always been powerful candidates for electrophysiological techniques, which enable studying a wide range of properties of membrane proteins including ion channels or receptors. A variety of approaches defined by the types of electrodes as well as recording apparatus are currently available [66]. The classic experimental approach used in conjunction with Xenopus oocytes is two-electrode voltage clamp (TEVC) (Figure 3). The method measures the small ionic currents through the membranes of excitable cells using two electrodes: one for injecting the current, and another for holding the voltage of the membrane. The injected current represents the sum of all ionic currents from the cell [67]. In voltage clamping, membrane potential of the oocyte is controlled and the following current response is recorded. Voltage clamping of ion channels is robust and well established and allows the study of the voltage-, ligand-, and time-dependence of channel openings and closings.

Figure 3:

Comparison of two-electrode voltage clamp (TEVC) and path clamping configurations. In TEVC, two separate electrodes are inserted into the target cell. The voltage sensing electrode measures the potential across the cell membrane. The current passing electrode, which is inserted into the center of the cell, delivers current to the target cell. The TEVC amplifier delivers current to the current passing electrode to maintain a specified potential at the cell membrane. The bath is held at earth ground. In some configurations, a driven ground can be used to manipulate the membrane potential. The voltage and current data are collected by analog-to-digital converter and analyzed on a computer. In patch clamping, the patch electrode is used to form a giga-ohm seal on the cell membrane. The bath is held at earth ground. Once the seal forms, the headstage measures the potential across the membrane patch and delivers current to maintain the specified potential of the membrane patch. Due to the size of the Xenopus oocyte, patch clamping will be limited to measuring ion channel currents in the membrane under the patch electrode. The ion channel currents could be measured in the inside-out or the outside-out configuration on the cell membrane. The voltage and current data are recorded by an analog-to-digital converter and analyzed on a computer.

In addition to TEVC, patch clamping is used to study individual ion channels and only controls the voltage of the membrane section attached to the tip of the micropipette (Figure 3). The patch clamping technique is much more laborious as it requires localizing the channel on a patch of cell membrane, and demands high precision recording instrumentation to measure very small currents, as well as high quality pipettes to minimize the background electrical noise (i.e. capacitive current). However, mastering this method and overcoming the technical hurdles provides invaluable information regarding the activity of single individual channels. Openings and closings of the ion channel, its modulation by endogenous and exogenous allosteric and orthosteric modulators, can be used to screen potential drugs.

One of the major requirements and challenges to performing patch clamping with oocytes is a clean and readily accessible surface that is protected by the vitelline membrane. This outer membrane composed from glycoproteins provides the oocyte’ structural rigidity and keeps the oocytes in a spherical shape. The vitelline membrane is usually removed either manually with forceps (most common, but also demanding fine motor skills) or with the collagenase treatment that dissolves the membrane [68]. Due to the high vulnerability of devitellinized oocytes, extreme care must be taken to handle the oocytes for electrophysiological recording.

Once the vitelline membrane is removed, the patch clamp can be performed. Several typical single channel configurations of patch clamp used in oocytes are shown in Figure 4. The difference between the methods lies in the relative orientation of the oocyte membrane and the rim of the glass electrode. Cell-attached configuration forms a good gigaseal with the intact membrane and is often used to study ligand-gated channels. A whole-cell patch clamping where the patch clamping electrode is used to attain direct electrical contact with the interior of the cell allows the entire cell membrane’s voltage to be controlled. This technique is applied more often on smaller cells, such as mammalian cells HEK 293 cells and is not optimal when used with cells the size of Xenopus oocytes [69], resulting in poor space voltage clamp regulation. The loose patch involves a comparatively low resistance seal and is also not very commonly used in oocytes. This would work for cells with high ion current density but would have large electrical noise characteristics.

Figure 4:

Typical configurations of patch clamp recording. Cell-attached patch: a patch electrode is attached to the cell but the membrane is not broken; Whole cell patch: A patch electrode is placed in a cell and the membrane is ruptured via suction. Loose patch: the patch electrode does not form a tight, gigaseal contact with the membrane; Outside-out: after forming a gigaseal and suction, the patch electrode is slowly withdrawn from the oocyte, leaving a portion of the membrane outside of the electrode; Inside-out: after forming a gigaseal and suction, the patch electrode is quickly withdrawn from the oocyte, leaving a portion of the membrane inside the electrode.

The other two techniques, outside-out and inside-out, are commonly used. For the outside-out patch configuration, once the seal forms the membrane in the pipette is ruptured, and the pipette is slowly withdrawn giving the membrane a chance to form a bleb at the tip of the pipette. The extracellular surface of the ion channels would be in their normal orientation giving the researcher the ability to apply extracellular ligands or alter saline composition. For the inside-out patch configuration, once the seal forms the patch can be ripped out of the membrane leaving the patch with its ion channels with the cytoplasmic side of the ion channel exposed to the bath. The inside-out configuration gives the researcher access to the cytoplasmic surface of the channel to activate ligand-gated channels or alter saline composition. The details of these systems are provided in the recent edition of The Axon Guide, which covers many electrophysiology and biophysics laboratory techniques [70].

3.2. High-throughput electrophysiology of oocytes for drug discovery

Typically, voltage clamping is used to screen the effects of drugs or mutated membrane proteins on a single ion channel or on a single cell, one cell at a time. While such electrophysiological measurements can provide high-resolution information about ion channel performance and functional state on a microsecond timescale, these techniques suffer from being labor-intensive and time-consuming. Indeed, the major tool for ion channel research, the patch-clamp technique, has used the same principle since it was described in 1981 [71]. (This classic paper introducing “giga-seal” has been cited in more than 19,000 publications). The situation changed in 1997, when the first commercial parallel multichannel patch-clamp instrumentation came to the market enabling true high throughput screening (HTS) approach in drug discovery [72]. Relatively large libraries of compounds were screened to develop better agonists or antagonists for epilepsy, major depressive disorder, and neuropathic pain [67, 69, 73, 74].

Multichannel patch recording, using automated technologies, generated a demand for large numbers of oocytes to be injected with ion channels, neurotransmitter transporters, and other genes. Traditional nanoliter injections are generally carried out manually, one cell at a time, leading to low throughput and weakly reproducible expressions of ion channels between different oocytes. Automated systems capable of injecting up to 600 oocytes per hour became available in early 2000’s [54]. With these technical advances, automated patch clamping systems and injection machines, recording large numbers of oocytes can be achieved within a short period of time resulting in HTS.

3.3. Voltage-clamp fluorometry (VCF)

Compared to the electrophysiological methods that remains a gold standard for understanding the mechanism of neurological disorders, fluorescence-based techniques are fairly new with the first developments of voltage-clamp fluorometry (VCF) in oocytes reported in 1996 [75]. In this original method, site-specific fluorescent labeling of a potassium channel protein with an environment-sensitive fluorophore was combined with voltage clamping to measure conformational rearrangement underlying potassium channel gating. The overall design of the instrumentation and procedure is straightforward (Figure 5, from [76]) . However it requires a strategy of careful cysteine site mutations of the ion channel mRNA, targeting the external exposed part of the chain, in conjunction with the silencing of the native cysteines, to increase the specificity of fluorophore labeling and reduction of background fluorescence [77]. Today, the fluorescence techniques encompass a number of methods that rely on dyes covalently bound to the specific subunits [78, 79], voltage sensitive probes [80], or from a great assortment of endogenously expressed fluorescent proteins. This fluorescence based technology rapidly moved from mechanistic studies to drug development. High specificity drugs are expected to induce conformational and other changes in the ion channels and transport proteins, leading to the alterations of the probes’ fluorescent properties (i.e. intensity, wavelength of emission, polarization, or lifetime). Using Alexa Fluor 546 conjugated to the ρ1 GABA_A receptor and electrophysiological techniques, Eaton et al. tested a library of neuroactive steroids and analogues to modulate human ρ1 GABA_A receptor function in the treatment of visual, sleep, and cognitive disorders [81].

Figure 5:

A) Potassium channel construct under resting (transparent) and activated (solid) states. The pink dot represents the engineered cysteine residue and yellow star denotes the tethered fluorophore. The movement of the S4 segment causes fluorophore displacement. As a result of quenching by surrounding residues, and changes in fluorophore environment, fluorescence emission is altered. B) mRNA encoding constructs are injected into Xenopus oocytes. Channels express at high levels after several days. Channels are then labeled with a fluorophore, which conjugates to the introduced cysteine residues. An example highlighting cut-open oocyte recording, which allows resolution of fast kinetics by clamping a small membrane patch. The upper chamber filled with external solution is clamped to the command voltages, while the bottom channel filled with internal solution is connected to ground. Both currents and fluorescence emission are recorded. With permission from Elsevier [76].

Further improvement to VCF, the cut-open vaseline gap configuration (COVG) [82] provides access to the cell interior thus enabling the measurement of internally fluorescently-labelled cysteines. This technique involves several vertically stacked solution chambers. After cutting a section of the plasma membrane, an electrical connection is created between the ooplasm and upper dish solution, and Vaseline is used to electronically isolate each chamber and the rest of the cell. The resulting setup provides less access resistance for intracellular current injection, rapid voltage steps, and greater membrane stability [73]. However, it is a much more complex technique than TEVC or patch clamp.

Alternatively, the access to the internal subunits can be realized through patch-clamp fluorometry (PCF) [83, 84], which combines fluorescence measurements and patch-clamp recordings. PCF also offers several advantages, such as better signal-to-noise ratio of fluorescence signals and high time resolution of current recordings [84]. Subsequently, PCF has been applied to many different ion channels, including those related to the drug discovery and screening. Another possibility would be TEVC with a third electrode loaded with the fluorescent probe. After labelling of the internal cysteine residues of interest, TEVC and fluorometry could be performed.

4. APPLICATIONS OF OOCYTES IN DRUG DISCOVERY

The origin of many neurological disorders lie in the mutations of ion channels. Specifically, epilepsy has been tied to over 150 different mutations in sodium channels that can lead to changes in subunit expression [13]. While many studies focus on the functional characteristics of these variant channels, Xenopus can also act as a system to study how changes in the genetic sequence can affect drug interactions. The oocyte’s ability to rapidly and easily express proteins has allowed researchers to compare drug effects on large libraries of mutants at relatively high throughput speeds. Perhaps more important than studying individual gene mutations, Xenopus oocytes have become a tool to identify the genetic causes of ion channel differences between species.

4.1. Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia, and it is characterized as a progressive degradation of cognitive functions starting with a decline of memory that eventually leads to complete dysfunction of the motor system [85]. The major hallmarks of AD include extracellular amyloid plaques and intracellular neurofibrillary tangles that accompany the loss of neurons, white matter, and synapses in the brain [86]. Some major approved drug treatments that improve cognitive function of AD victims include cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists [87]. Of these drugs that have been approved to treat AD, none of them have been able to alter the course of the disease, and instead, current drug treatments can only act on the symptoms of the disease [88].

Parodi et al. have demonstrated that Xenopus oocytes respond to amyloid aggregates similarly to neurons and have the potential to become experimental models to study the electrical changes caused by these amyloid aggregates [89]. As previously stated, ion channels from diseased human brain samples have been successfully micro-transplanted into the oocyte plasma membrane. This membrane microtransplantation technique was pioneered by Miledi’s lab in 1995 to examine the possibility of transplantation of Torpedo electroplaque membranes that had functioning ion channels and receptors [62, 90]. Since then, several other groups have refined the methodology [63, 91]. Currently, researchers are able to selectively target synaptic receptors and ion channels in synaptosomes extracted from total brain membranes for micro-transplantation, and these transplanted synaptosomes provide more reliable current measurements as well as a more focused view into the ionic mechanisms of AD [92].

In order to develop high potency, disease-treating drugs, researchers need a better understanding of the mechanisms of AD. Xenopus oocytes have played a major role in this understanding and ultimately expanded the number of validated drug targets. One of the central hypotheses for the origin of AD is that the amyloid plaques disrupt neuronal cell membranes and create pores that majorly affect ionic homeostasis [93]. The amyloid fibers allosterically bind to α7 nicotinic acetylcholine receptor (α7 nAChR), a ligand-gated Ca²⁺-permeable ion channel that has long been known to play an important role in cognitive function [94]. Co-localization and binding of amyloid fiberes with α7 nAChR induces neuron apoptosis and reduces α7 nAChR expression. Prevention of this binding of amyloid with the α7 nAChR by using agonists for α7 nAChR resulted in the protection of the neuron and significantly improved the learning and memory ability of the animal models. That makes α7 nAChR an attractive pharmacological target for AD as well as other neurological and psychiatric disorders [95]. Although many possible targets besides α7 have been discovered for AD, we will limit our discussion to α7 since this target attracted a large activity among oocyte community.

Using Xenopus oocytes, a number of active compounds were identified that selectively target α7 nAChR. Selected agonists for α7 nAChR that advanced to clinical studies include GTS-21 from the University of Florida and Taiho Pharmaceutical [96], ABT-126 from AbbVie [97], TC-5619 developed by Tagacept [98], and recently encenicline from Forum Pharmaceuticals [28], and tropisetron, a drug currently approved for treatment of chemotherapy induced nausea and vomiting [99]. Although these compounds have yet to be approved for AD, the strong effort to selectively target α7 nAChR with minimum side effects has the potential for future discovery.

The affinity gap between receptors of different species is highly problematic when considering that most drug discovery studies depend on using rodent or other animal models. In Xenopus oocytes, however, a strong effort has been focused on comparing human and rat channel homologues and identifying key genetic sequences that are responsible for the interspecies affinity gap. The α7 nAChR receptor is highly conserved between humans and rats, differing by only 10 of 208 amino acid residues [100]. This small difference, however, was found to be responsible for a 1.5–2 times drop in drug affinity [100]. The authors of this report were able to create 10 single-residue mutants for each of the differing sites and used a Xenopus expression system to determine the key residue responsible for the difference in drug affinity. Furthermore, the identified residue mutation was found to fix homologous receptor affinity gaps for a number of other drugs.

4.2. Amyotrophic Lateral Sclerosis (ALS)

ALS is a progressive, neurodegenerative disease, affecting motor neurons. Symptomatically, it presents as a gradual paralysis that eventually leads to the death of the patent. There is currently only one approved treatment that is mildly therapeutic, Riluzole (Sanofi Aventis), which first received authorization in 1995 [101]. Numerous other compounds have been investigated; however, they mostly failed to pass clinical trials. Outside of Riluzole, there are two promising treatments, oral masitinib and intravenous edaravone [101]. All of these compounds, however, can only improve the quality of life for the patient as there is no cure for ALS. The difficulty in curing ALS is partly that the cause is not known in more than 90% of cases and may be due to many factors that are in play. These factors include oxidative stress, protein aggregation, impairment of axonal transport, neurotransmitter imbalances, and neuroinflammation [102]. The hallmark feature of ALS is the death of motor neurons in the motor cortex of the brain, brainstem and spinal cord apparently due to the presence of abnormal aggregations of protein in the cytoplasm of motor neurons. Additionally, despite being considered a CNS disease, there has been new evidence that defects in skeletal muscle leading to muscle denervation precedes neuronal death and plays a major role in the pathogenesis of ALS [103].

Xenopus oocytes have been useful in isolating and characterizing the role of skeletal muscle in the onset of ALS. Also, these models help circumvent many of the technical difficulties and ethical concerns of studying ALS patients. In particular, Palma et al. have been able to investigate acetylcholine receptors by micro-transplantation of muscle membranes collected from ALS patients via needle biopsy [104]. Their findings show that acetylcholine receptors from ALS patients show a significant decrease in acetylcholine affinity, which may be related to the clinical symptoms of muscle paralysis. These results suggest that muscle acetylcholine receptors are a potential new target for ALS drug development and have triggered a series of studies to further investigate the role of skeletal muscle in ALS pathogenesis. One of these studies investigates the role of palmitoylethanolamide (PEA) which was been previously shown to improve the clinical condition of one ALS patient [105]. In the study, the authors treated Xenopus oocytes with PEA after diseased muscle acetylcholine receptors had been micro-transplanted into the oocyte membrane. They found that PEA reduced the desensitization of acetylcholine receptors in ALS oocytes. Furthermore, the authors identified that PEA also delayed the respiratory impairment of a selected group of ALS patients [106]. These results are the first demonstration of a compound that can affect skeletal muscle function in ALS at both the molecular level and in clinical practice.

4.3. Chronic pain and neuropathies

Overuse of the opioid drugs to treat pain recently turned into the center of attention in many developed countries. Due to the high risk of addiction, the opioid epidemic in the U.S. has become a national emergency that requires an immediate solution in the form of non-addictive drugs with no other side effects [107]. In that regard, Xenopus oocytes are frequently used as models for expressing proteins related to chronic pain and the search for new drugs. Thus, the search for more selective compounds to treat chronic pain has led to the study of a synthetic peptide PnPP-19 that resemble the active part of the known Phoneutria nigriventer spider toxin [108]. In this study, a variety of opioid receptors (i.e. human μ opioid receptor (hMOR), human k opioid receptor (hKOR), and human d opioid receptor (hDOR)) as well as ion channels (i.e. GIRK1, GIRK2) expressed in Xenopus oocytes were tested for their peptide specificity.

Other noticeable examples that involved oocytes for pain research include the measurements of GABA_A receptors functions toward understanding migraine and drug development for migraine treatment [109]. The drugs targeting GABA ion-channels appear as a promising avenue for both the acute treatment of migraine and migraine prevention [110]. A number of GABA subunits represent the major inhibitory neurotransmitter receptors in the mammalian brain. Poor performance of the GABA receptors appear to be responsible for the chronic migraine [110]. Recently, a new class of deuterated ligands functionally selective for GABA_A α6 subtype with improved metabolic stability and enhanced bioavailability was discovered for treatment of migraine using recombinant GABA_A receptor subtypes expressed in Xenopus oocytes [111].

The search for new pain-related targets led to the ligand-gated cation channel microglial P2X7 purinergic receptors [112]. P2X7 purinergic receptors are broadly expressed in peripheral nervous system, central nervous system, and immune system, which mediate and modulate pain. Mice with a surgically induced chronic neuropathy (in a partial nerve ligation model) that lack this receptor showed complete suppression of the hypersensitivity to mechanical and thermal stimuli, while having normal nociceptive responses [113]. Drugs which block this target may have the potential to deliver broad-spectrum analgesia and relief from the chronic pain. Xenopus oocytes transfected with human P2X7 purinergic receptors were shown to be selectively inhibited by colchicine [114] and recently by a commonly used mild anesthetic Lidocaine [115] thus opening up potentially new directions toward the treatment of the chronic pain.

In addition, a new type of pain caused by common by chemotherapy drugs leads to the condition known as chemotherapy induced peripheral neuropathy (CIPN). CIPN occurs in nearly 10 – 60% of patients treated with first-line chemotherapy drugs. Patients with acute neurotoxicity appear to be at increased risk for chronic neuropathy in which painful symptoms persist long after cessation of chemotherapy. Deterioration of sensory neurons and their axons leads to the loss of nerve fiber density and nociception, and this also puts the patient at greater risk of serious damage to the hands or feet. CIPN can be debilitating and is frequently the primary reason for patients choosing to discontinue chemotherapy. To date, most of the CIPN prevention trials have not demonstrated benefits compared to treatment with a placebo, and therefore novel targets are critically needed. A number of recent studies used Xenopus oocytes to test new targets and identify new active drug candidates. Thus, Romero et al. developed a novel peptide RgIA4 that exhibits high potency for both human and rodent α9α10 nAChR [116], a recently discovered ion channel that has been implicated in the neuropathic pain, including CIPN [117].

4.4. Limitations of oocytes

Like every in vitro model, Xenopus oocytes have drawbacks that must be considered before selection. Some of these limitations have been mentioned in the Introduction. Of others, the most critical is the oocytes exhibit limited longevity, with most oocytes surviving only 10–15 days post-harvest before significantly deteriorating in quality. One factor that contributes to oocyte lifespan, and quality are seasonal variation. It has been observed that, depending on the time of year, oocyte longevity and quality can be severely affected and even prevent the use in experiments [118, 119]. Despite this issue, a majority of solutions that increase oocyte lifespan are in the form of anecdotal advice, and there are very few examples of literature offering empirically-backed solutions. As a result, oocytes are typically treated as one-time use cells and are frequently discarded. With the current price of $2 per a single high quality oocyte (from Ecocyte Bioscience LLC), the HTS studies that involve oocytes might become costly. To decrease the cost, especially for large scale studies, a frog colony can be maintained. The cost of frogs is inexpensive, around $26 for a female and $20 for a male frog. However handling frogs requires an appropriately equipped animal facility and costs associated with breeding and animal housing as well as surgical skills [57]. Moving forward, developing storage protocols to increase the lifespan of Xenopus oocytes would greatly enhance their value as an in vitro model as well as cut down on the number of oocytes harvested for experimentation.

Immature Xenopus oocytes possess several endogenous ionic conductances that will need to be accounted for in the design of the experiments involving injection of mRNA for ion channels or receptors The list summarized by Dascal in his comprehensive review [120] includes calcium dependent chloride current, voltage-activated calcium dependent chloride current, voltage-dependent calcium current, hyperpolarization-evoked chloride current, slow potassium and slow voltage-dependent sodium currents, muscarinic acetylcholine evoked chloride current, epinephrine and dopamine evoked potassium currents. In particular, exogenous ion currents that involve calcium currents will also activate the endogenous calcium-dependent chloride current, confounding the results of the measurements. This kind of interaction of current measurements due to the flow of calcium was observed with the currents measured from activation of mammalian alpha-7 nicotinic acetylcholine receptors from injection of mRNA into frog oocytes [121] minimized the effect of the endogenous calcium dependent chloride current by substituting calcium with barium while maintaining the divalency needed by the nicotinic acetylcholine receptor to properly function.

Another common limitation comes from the measurements of the ionic current. When performing two-electrode voltage clamp (TEVC), the large size of the oocyte can become a drawback as it can lead to sources of large capacitive artifacts in the electrophysiological recording. This artifact from cell capacitance can be corrected today with proper amplifier design. For drug discovery, the most serious limitation has been relatively low throughput. Even using the latest technological advances in HTS, only small focused libraries, tens and hundreds of compounds, can be screened in a reasonable time frame compare to a tens of thousands in a typical pharma screening. All of that contributes to the low number of new drugs, albeit the sale of each drug is in general quite high, and low novel treatable disease targets. This also explains a shrinking number of publications using Xenopus oocytes from >900 papers in the year 2000 to about 300 in the year 2018 (based on the Pubmed database).

5. CONCLUSIONS

Neurological diseases present a difficult challenge in drug discovery. Many of the current treatments have limited efficiency or result in a variety of debilitating side effects. The search of new therapies is of a paramount importance, since the number of patients that require a better treatment is growing rapidly. As an in vitro model, Xenopus oocytes provide the drug developer with many distinct advantages, including size, durability, and efficiency in exogenous protein expression. In terms of drug discovery, these oocytes remain the gold standard for high-precision testing of the drugs on single membrane components to target specific receptors whether the receptor is an individual ion channel or the combination of ion channels with regulating proteins and other receptors. To facilitate the transition to clinical trials, many advances in the field of oocytes today include the expression of human ion channels and transplantation of oocytes with human tissues. These systems in conjunction with high-throughput methods, although limited to relatively small libraries, has enabled the identification of many approved drugs and drugs that are currently in the clinical trials. Although our review reflected only a few types of neurological disorders such as AD, ALS, and chronic pain, many other neurodegenerative and psychiatric diseases benefit from oocyte studies as the first step to model the disease. Thus Xenopus oocytes continue to be used as a reliable source of high resolution information and remain part of the mainstream of electrophysiological studies to characterize mutant ion channels and identify new drugs.

6. EXPERT OPINION

A recent analysis conducted in 2014 estimated annual cost to the US of nine of the most common neurological diseases at almost $800 billion dollars [1]. These conditions include Alzheimer’s disease and other dementias, epilepsy, multiple sclerosis, spinal cord injury, low back pain, stroke, traumatic brain injury, migraine, and Parkinson’s disease. Even without accounting other and rare neurological and peripheral nerve disorders such as CIPN, diabetic neuropathy, and ALS, the overall burden is astounding. As the authors of this report noticed, this number will rapidly increase in the coming years due to an aging population since a neurological disease is so much more prevalent in the elderly. While many diseases such as cardiac, infectious, and even cancer can be prevented by regular screening, early diagnostics, diet, and physical activity, neurological diseases, with rare exceptions, cannot be prevented this way and as a result are more challenging to treat.

As the response to this problem National Institutes of Health (USA) has issued several programs in the last few years to establish more fundamental understanding of the neurological diseases and identify cures. Examples include the BRAIN initiative, National Pain Strategy (NPS), and Stimulating Peripheral Activity to Relieve Conditions (SPARC). These initiatives provide an action plan for reducing the neurological burden through investment in the infrastructure for neuroscience research and enhanced clinical management of neurological disorders.

From the perspective of drug discovery, the investment in this research will bring new molecular targets, where oocytes will play their traditionally important role in target validation and drug testing. As treatments and cures for previously terminal diseases are discovered, there will be an emphasis on tackling the emerging diseases that either arise as side effects from those treatments or as complications associated with aging. While many of these neuropathologies are multi-faceted, the development of pharmaceutical treatments has the ability to majorly improve patient symptoms or even cure the disease in question.

The ability for the investigators to take ion channels or receptors from frozen human tissue samples and transplant them into a Xenopus oocyte has greatly expanded the opportunities to study neurological disease. This technique takes advantage of the oocytes amenability to microinjection and exogenous proteins. It also provides the chance to directly study the exact receptors that were the source of neurological disease, which improves the translatability of the results. Additionally, the technique has been refined to allow the transplantation of specific synaptic receptors or channels. This not only allows researchers to understand the functional characteristics of native disease-causing membrane proteins but also provides the ability for the gathering of data personalized to an individual. Currently, the number of neurological diseases that this micro-transplantation has been applied to is limited, but moving forward, represents an area with a great promise for the study of other diseases. Already, ion channels have been collected from samples of skeletal muscle, which demonstrates the applicability of this technique to tissues outside of the brain.

Xenopus oocytes have been tremendously valuable for its robustness, ease of use, and ability to reliably express exogenous membrane proteins. As Xenopus oocytes continue to improve as models, their role in drug discovery for neurological diseases will only expand. With the development of higher throughput electrophysiological methods in combination with all the other mentioned advancements, these oocytes have the ability to rapidly and accurately screen through large libraries of molecules and aid in the development of therapeutic drugs.