New approaches to epilepsy treatment: optogenetics and DREADDs

Epilepsy refers to a group of disorders that are characterized by recurrent seizures. It is a relatively common neurological condition, and is considered the most common serious (implying that there is a risk of mortality) brain disorder, affecting around 2.2 million Americans.

Watch this 2-Minute Neuroscience video to learn more about epilepsy.

The seizures associated with epilepsy are not homogenous; they can have a drastically different presentation depending on the patient, the part of the brain the seizure originates in, and how much of the brain the seizure affects. For example, seizures can involve clonic activity (i.e. jerking movements), tonic activity (i.e. rigid contraction of muscles), atonia (i.e. loss of muscle activity), or any combination of motor movements and/or loss of motor activity. On the other hand, they can simply be associated with a brief and subtle loss of consciousness, as in the case of an absence seizure.

One attribute that all seizures have in common, however, is excessive neural activity. Seizures are generally characterized by an increased rate of firing in a population of neurons and/or synchronous firing (i.e. neurons that are normally activated at disparate times are all firing together, leading to large spikes in neural activity) by a neuronal population. Because seizures involve an excessive level of neural activity, ictogenesis (i.e. the generation of seizures) has commonly been considered to involve either the direct excitation of neurons or the failure of a mechanism that inhibits the excitation of neurons.

Pharmacological treatments for epilepsy have been designed from this perspective, and have generally involved drugs that either decrease neural activation or increase neural inhibition. For example, drugs like carbamazepine and lamotrigine treat epilepsy by reducing activity at sodium channels in neurons, which makes neurons less likely to fire action potentials and leads to less overall neuronal activity. Other drugs, like phenobarbital and lorazepam, actively promote neural inhibition by increasing the stimulation of gamma-aminobutyric acid (GABA) receptors. GABA receptor activation typically makes neurons less likely to fire, which also reduces overall neural activity.

Pharmacological treatments for epilepsy, however, leave much to be desired. The side effects associated with them range from minor (e.g. fatigue) to serious (e.g. liver failure), and about 30% of epilepsy cases don't even respond to current pharmacological treatments. Surgical options (e.g. removing an area of brain tissue where seizures originate) can be considered in severe cases. Clearly, however, this is an irreversible treatment and also one that lacks specificity, which means that some potentially harmless (but important) brain tissue is likely to be removed along with areas from where seizures are emerging. Although surgical procedures can allow about 60-70% of patients to be seizure free within a year after the procedure, after 10 years more than half of patients begin to experience seizures again.

One of the major limitations to the current approaches to treating epilepsy is that they lack specificity. For, even if seizure activity can be traced back to excess neural excitation or deficiencies in neural inhibition, it is clear that these problems are not occurring all of the time because--except in rare cases--seizures are intermittent and represent only a small percentage of overall brain activity. Drugs that increase inhibition or reduce excitation, however, are having these effects continually (as is surgery, of course). Thus, the treatment of epilepsy is a rather crude approach that involves exerting a constant effect on the brain in the hopes of preventing a relatively rare event.

Because of this, efforts at designing new treatments for epilepsy have focused on more selective techniques. Although these approaches--which hypothetically involve targeting only neurons involved in ictogenesis--are still a long way from being used in humans, there is some promise associated with them. One method, optogenetics, targets seizure activity by incorporating light-sensitive proteins into neurons and then controlling their activity with the application of light. Another approach, designer receptors exclusively activated by designer drugs (DREADDs), focuses on ictogenesis by incorporating genetically engineered receptors that only respond to a specific ligand into neurons and then controlling neuronal activity through the administration of that ligand.

Optogenetics for the treatment of epilepsy

Optogenetics is a field that combines insights from optics and genetics to manipulate the activity of neurons. It generally involves the use of gene therapy techniques to promote the expression of genes that encode for light sensitive proteins called opsins. In most cases, genes for opsins are carried into an organism after being incorporated into a virus' DNA (the virus in this case is known as a viral vector) or an animal is genetically engineered to express opsin genes in certain neurons from birth. Opsin expression can be targeted to specific cell types and the proteins can be used to create receptors or ion channels that are sensitive to light. When light is delivered to these neurons--either via an optical fiber inserted into the brain or with newer technologies that allow wireless external delivery of light--the opsins are activated. This allows exposure to light to act like an on-off switch for the neurons in which opsins are expressed, making them suitable for experimental or therapeutic manipulation.

One way optogenetics can be used as a treatment for epilepsy is by promoting the expression of a light-sensitive ion channel that, when activated, allows a flow of negatively charged chloride ions into the cell. This hyperpolarizes the neuron and makes it less likely to fire an action potential. Alternatively, opsin ion channels can be expressed on GABA neurons that, when activated, cause increased GABA activity and thus promote general neuronal inhibition. Both of these approaches lead to reduced activity of neuronal populations, potentially decreasing the excessive activity associated with seizures.

When linked to some method of seizure detection, optogenetics can be used to inhibit seizure activity at the first indication of its occurrence. This has already been achieved in experimental animals. For example, Krook-Magnuson et al. (2012) promoted either the expression of inhibitory channel opsins or opsins that activate GABA neurons in different groups of mice, then monitored seizure activity using electroencephalography (EEG) after the injection of a substance that promotes seizures. When seizure activity was detected on the EEG, it automatically triggered the application of light to activate the opsins. In both groups (inhibitory channel opsins and excitatory opsins on GABA neurons), light application rapidly stopped seizures.

Thus, when combined with seizure activity monitoring, optogenetics provides a way to selectively control neural excitation, cutting seizures off as soon as they begin. Optogenetics is still a relatively new field, however, and the work in this area has not yet translated into clinical approaches with humans. There are some significant hurdles to overcome before that can happen. One involves the need for a device that can non-invasively and effectively deliver light, another concerns the need for a non-stationary device that can monitor seizure activity. Advances in wireless light delivery, however, have already been made, and an implantable device to monitor seizure activity in humans was recently tested for the first time. Therefore, while this technology is not ready to be applied to epilepsy treatment in humans yet, its use is feasible in the not-so-distant future.

DREADDs for the treatment of epilepsy

Designer receptors exclusively activated by designer drugs, or DREADDs, are another approach that addresses the desire for specificity in epilepsy treatment. The use of DREADDs involves the manipulation of genes that encode for neurotransmitter receptors, then the forced expression of those mutated genes in an experimental animal. Receptors can be engineered so they no longer respond to their natural ligand, but instead only respond to a synthetic, exogenously administered drug. DREADD expression can be targeted to specific cell populations and, like the optogenetic methods discussed above, the receptors can be used to activate inhibitory neurons or inhibit excitatory neurons.

For example, Katzel et al. (2014) modified an inhibitory muscarinic acetylcholine receptor to no longer respond to acetylcholine but instead only to a synthetic ligand called clozapine-N-oxide (CNO); they then promoted the expression of this receptor in the motor cortices of rats. They administered a seizure-causing substance, then administered CNO, and found that CNO administration significantly reduced seizure activity.

Therefore, it seems that DREADDs also have potential for the targeted treatment of epilepsy. Because activation of DREADDs only requires taking a pill, it is considered less invasive than current optogenetic approaches. However, optogenetics possesses greater temporal specificity in that it can be activated immediately upon the onset of seizure activity and terminated just as quickly. Synthetic ligands for DREADDs, on the other hand, must be administered in advance of ictogenesis to ensure the drug is available to inhibit seizure activity when it begins, and will remain active in a patient's system until the drug is metabolized by the body.

Just as with optogenetics, though, there are some hurdles that must be overcome for DREADD use to translate into the clinical treatment of epilepsy. For example, individuals tend to vary considerably in how quickly they metabolize drugs. Thus, there might be some variation in the time span of protection offered by administration of a DREADD ligand, which in the case of potentially severe seizures could be dangerous. Also, although the ligands used for DREADD activation are chosen based on their selectivity for the designer receptor, a metabolite of CNO is clozapine, a commonly-used antipsychotic drug that also activates other receptors. In rodents, this did not translate into side effects, but the potential for metabolites of synthetic ligands to be biologically active must be considered when attempting to apply the technology to human populations. 

Optogenetics and DREADDs both represent intriguing approaches to treating epilepsy in the future. The intrigue stems primarily from their ability to only target certain cells, which is likely to reduce the occurrence of side effects. Regardless, even if these technologies aren't able to be used to treat humans for a long time--or ever--they still have a place in epilepsy research. For, the use of these tools also allows us more control over seizures in experimental animals, which makes a more thorough dissection of the seizure process possible. At the very least, this should provide more insight into a dangerous, yet relatively common, neurological disorder.

Krook-Magnuson, E., & Soltesz, I. (2015). Beyond the hammer and the scalpel: selective circuit control for the epilepsies Nature Neuroscience, 18 (3), 331-338 DOI: 10.1038/nn.3943

Gene Therapy for Prion Diseases

Prion diseases are relatively rare in humans. The most common, Creutzfeldt-Jakob disease (CJD), afflicts only about one in every million people. Despite their low prevalence, however, these diseases (also known as transmissible spongiform encephalopathies, or TSEs) receive a fair amount of attention from the media and the scientific community. This interest is probably due to their enigmatic mechanism, potential for epidemic spreading, frightening neurodegenerative features, and (as of yet) incurability.

TSEs are neurodegenerative diseases thought to be the result of a prion infection. This distinguishes them from most other sicknesses, which are caused by microbial infections. Prions are infectious agents made up entirely of proteins (the word itself comes from a combination of proteinaceous and infectious).

A prion protein called PrPC (the C stands for cellular) is commonly present on the membranes of our cells, although its function has not yet been fully resolved. PrPSc (the Sc is for Scrapie, the first identified prion disease—in sheep) is an isoform of PrPC and the toxic form of PrP. When it enters the brain it can cause conformational changes in PrPC, turning it into PrPSc.

PrPSc is extremely resistant to being broken down. Thus, it accumulates in the brain, forming protein aggregates known as amyloid fibers. These are toxic to brain cells, and eventually kill them. Astrocytes, which perform a number of supporting functions in the cell (one of which is cleaning up), find the dead neurons and digest them.

This creates actual holes in the brain, giving it a sponge-like appearance (and the reason for these disorders being referred to as spongiform). This continued neurodegeneration leads to a number of clinical symptoms, like changes in personality, depression, involuntary movements, lack of coordination, dementia, and eventually the complete loss of the ability to move or speak. TSEs are currently incurable, and an effective method of therapeutic treatment has not been found. The aggregation of PrPScs occurs over a long period of time, giving the diseases incubation periods that range from 10-60 years depending on the disease type.

TSEs can be the result of genetic or sporadic (non-genetic) causes. A mutation in the prion protein (PRNP) gene can cause the production of PrPSc instead of PrPC, leading to a prion disease. TSEs are also contagious—not through the air or normal contact, but through exposure to infected tissue, body fluids, or contaminated medical instruments (due to the durability of prions, they can survive normal sterilization procedures).

Unfortunately, we have learned about how TSEs are spread by witnessing several deadly epidemics. Around the middle of the twentieth century, a TSE arose in a New Guinean tribal people called the Fore. It is thought to have spread through cannibalistic ritual practices, and killed over 1,000 of their people. In the 1980s 60 deaths were linked to the transmission of CJD through contaminated medical instruments. Around the same time, 85 people died after receiving prion-infected growth hormone injections. In the 1990s, a type of CJD called variant CJD (vCJD) was linked to eating beef infected with the bovine form of TSE, bovine spongiform encephalitis (BSE), or mad cow’s disease. vCJD has a shorter incubation period than CJD, with the median age at death being 28, versus 68 for CJD. The illness also has a longer duration, with a median of 15 months for vCJD and only 4-5 months for CJD. Up to 200 people worldwide have died from vCJD.

BSE is thought to be caused by feeding cattle the remains of other infected cattle. This practice was stopped in 1989. Due to the long incubation period of the disease, however, some fear that the real mad cow disease epidemic has yet to manifest itself.

An article in PloS One this month addresses a possible way to control such an outbreak, with the successful application of a gene therapy treatment for TSEs. A natural resistance to prion diseases has been discovered in both animals and humans, and specific mutant forms of the mouse Prnp gene have been found to reduce the replication of prions in infected cells.

The researchers involved in the study injected this mutant gene into the brains of mice infected with prions. In order to make the study more relevant to human TSEs, they did this during late stages of the disease, at 80 and 95 days post infection. This increases relevance because, due to the long incubation period of TSEs, most people are unaware they have contracted them until serious symptoms develop.

They found that, after two injections, treated mice survived 20% longer than non-treated mice. They exhibited substantial improvements in behavioral symptoms, as well as a significant reduction of spongiosis and astrocytic activity in the brain.

The authors suggest this effect occurred because the mutated Prpn gene produces a protein that cannot be converted into PrPSc. Additionally, the protein it makes competes with PrPC for PrPSc, slowing the conversion of existing PrPC to the toxic form. Basically, this means that the PrPSc doesn’t realize the new proteins can’t be transformed, and still attaches itself to them. This delays the overall disease progression, as many of these PrPScs are busy trying to make conformational changes to no avail.

These results are promising not only because they slow down the aggregation of toxic prions, but because the effect was demonstrated at such a late stage of disease. Unfortunately, the disease was slowed but not cured. Regardless, the hint of a successful method of treatment for prion diseases might be comforting to nervous meat eaters who are fearing a future vCJD outbreak. I’m a vegetarian (and have been for a long time), so as long as the soybeans in my tofu weren’t grown with meat and bone meal fertilizer, I feel reasonably safe.


Karine Toupet, Valérie Compan, Carole Crozet, Chantal Mourton-Gilles, Nadine Mestre-Francés, Françoise Ibos, Pierre Corbeau, Jean-Michel Verdier, Véronique Perrier, Alfred Lewin (2008). Effective Gene Therapy in a Mouse Model of Prion Diseases. PLoS ONE, 3 (7), 0- DOI:10.1371/journal.pone.0002773

Gene Therapy: Struggling to Leave the Past Behind

Gene therapy is a relatively new method of treatment that involves replacing the defective allele of a gene with a functional one. The technique, originally thought to hold great potential for the treatment of genetic diseases, was at first greeted with excitement and enthusiasm. This enthusiasm continued to grow after the first successful administration of gene therapy in 1990, to improve the health of four-year old Ashanthi Desilva (born with severe combined immunodeficiency).

Since then, however, gene therapy has had its ups and downs, hitting rock bottom with the death of 19-year old Jesse Gelsinger in 1990. Gelsinger wasn’t in a life or death situation. He volunteered for the study because of a brush with death he had early in life due to a genetically inherited liver disease. He volunteered with the hopes that a cure would relieve others from suffering through some of the trials he had as a young boy. Gelsinger, however, wasn’t informed of some of the possible dangers of the treatment he was about to undergo—dangers that the scientists involved in the study were cognizant of. They neglected to tell him, and he died several days after treatment.

Since then, gene therapy has struggled to creep out from under the shadow of that dark incident. Continued successes, however, indicate that gene therapy may still have the opportunity to live up to its once heralded potential. One example is a study reported this week in the New England Journal of Medicine that describes successfully using gene therapy to restore vision in three young adults born with severe blindness.

The subjects suffer from a disease known as Leber congenital amaurosis (LCA), which usually leads to complete blindness by middle age and is thought to be caused by a mutation in a gene called retinal pigment epithelium 65 (RPE65). The gene encodes for a protein that converts vitamin A into a form that can be used by the rods and cones of the eye to make rhodopsin (a pigment that absorbs light).

The researchers injected one eye of each patient with a harmless virus carrying the healthy form of the RPE65 gene. After only two weeks, all of the participants reported improved vision in dimly lit environments. Within six weeks, some of the patients were able to read several lines of an eye chart or navigate an obstacle course—dramatic improvements over their previous levels of legal blindness. The researchers involved suggest that, due to the efficacy of this treatment, it could eventually be applied to other eye disorders, such as macular degeneration.

Every advance made in the use of gene therapy is a major one, as after the death of Jesse Gelsinger, many were quick to condemn the use of the procedure as unsafe and irresponsible. While the scientists involved in the Gelsinger debacle deserve those criticisms, the procedure itself holds great promise for understanding and ameliorating some of the worst afflictions humans face. Hopefully one day the number of lives improved and saved through the use of gene therapy will soften the sting of the egregious mistakes made in its early history.

Using Gene Therapy to Treat Addiction

When you take into consideration the number of ways in which it can manifest itself, addiction is probably the most prevalent mental disorder that we don’t yet have a pharmaceutical treatment for. By identifying common neurobiological substrates that underlie all types of addiction, however, scientists hope to find drug targets that may one day allow it to be treated at least as methodically as other widespread disorders like depression. One of these commonalities found in the brains of addicted subjects involves a reduction of the number of available dopamine receptors in reward areas of the brain. Specifically, the density of a receptor called the D2 receptor is found to be decreased.

This diminished D2 receptor density can lead to addiction in two ways. A normal level of D2 receptors in an area of the brain called the nucleus accumbens is thought to play an important role in regulating impulsivity, and thus in avoiding an initial exposure to large amounts of drugs (or any addictive substance). Low levels may predispose drug users not only to trying drugs, but also to using them in amounts large enough to lead to addiction.

When a drug is used, it increases dopamine transmission. Over a period of time, this increase in dopamine activity in the brain can cause D2 receptors throughout the reward system to become depleted. This occurs due to a process called downregulation, which is a natural response of the body to decrease the number of receptors for a substance if the substance seems to be available in excessive amounts. Downregulation can lead to a disruption of the reward system that causes an addict to have difficulty finding pleasure in “normal” activities. The drug is the only substance that can facilitate a sense of pleasure, and compulsive use becomes difficult to avoid.

This understanding of the role of D2 receptors in addiction led researchers at the U.S. Department of Energy’s Brookhaven National Laboratory to search for a way to increase the number of D2 receptors in an addicted brain, in the hopes that doing so could reverse the process of addiction. They found gene therapy to be a successful way to do it.

First, the scientists trained a group of rats to self-administer cocaine (not a difficult thing to do, as most organisms tend to like cocaine). They then injected an innocuous virus that carried the gene for D2 receptor production into the rats’ brains. It was hoped the virus would insert the D2 receptor gene into the cells of the rats’ reward system, leading to an increase in D2 production, and a consequent reduction in addictive behavior.

After the gene therapy treatment, the rats showed a 75 percent decrease in cocaine self-administration. The change lasted for about six days before the rats returned to their previous usage. The group at Brookhaven has conducted a comparable experiment in the past with alcohol-addicted rats, and found similar results.

D2 receptor density has been shown to play a role in predisposition to addiction, as well as in the propagation of addictive behavior after its onset. With experimental results like those seen at Brookhaven, it appears manipulating D2 receptor density may have potentially beneficial effects in treating addiction. Unfortunately, given gene therapy’s checkered past (see here), it will be quite some time before such methods become available for use with humans. Primate studies, however, will most likely be the next step, and if all goes well perhaps gene therapy may one day end up being able to treat what could arguably be called the greatest behavioral scourge of our species: the repetitive pursuit of rewards long after they’ve lost their rewarding value, a.k.a. addiction.

Why Some People Didn't Give Up on Gene Therapy

Gene therapy, a treatment for disease that involves the insertion of healthy or disease-fighting genes into a person's cells, has undergone something of a roller coaster ride of public approval. Although hotly contested from its inception due to suggested ethical and methodological flaws, its first use in 1990 on four-year old Ashanti DeSilva to alleviate the symptoms of a rare immune disorder seemed successful. The next decade, however, instead of being one of vindication for gene therapy advocates, was fraught with disappointment due both to technical problems with its application, and to grossly unethical handling of its use in people.

In 1999, Jesse Gelsinger, an eighteen-year old with a genetically inherited liver disease, died in a clinical trial for gene therapy. Subsequent investigations found that the researchers involved in the study acted unscrupulously in a number of ways, such as not reporting serious side effects experienced by other patients and not disclosing that monkeys had died after similar treatment in previous studies. This provided more ammunition for opponents of gene therapy. Then, in a clinical trial in the beginning of this decade two children developed leukemia-like symptoms, leading to a temporary ban on clinical trials for the method.

Clinical trials were resumed, but as you can see scientists studying gene therapy have had many obstacles to overcome in getting people to focus on the potential of the treatment, and the successes it has had. Thus, news coming from researchers at the Cedars-Sinai Medical Center will likely be reason for gene therapy proponents to celebrate.

The group recently completed a study using gene therapy in rats to attack glioblastoma (GBM), the most common type of brain cancer. GBM is especially lethal, with a prognosis of six to twelve months of life after diagnosis. It is also extremely hard to treat, as, by the time it is diagnosed it has usually spread to other brain areas, making it difficult to surgically remove a tumor without leaving cancer cells behind. The blood-brain barrier, a safety mechanism that prevents toxins in the blood from entering the brain, stops chemotherapeutic agents from getting to tumor cells in effective amounts. Dendritic cells, which are essential to the immune process as they present foreign antigens on their cell surface to stimulate the immune response, do not naturally occur in the brain. Thus, the tumor in GBM grows unchecked by the immune system. This growth also usually causes behavioral changes and cognitive deficits as the tumor affects other areas of the brain.

The scientists at Cedars-Sinai, using a viral vector (a virus used as a vehicle to carry genetic material), sent two proteins to the cancer cells in the rats’ brains. One of the proteins attracted dendritic cells to the brain, which resulted in an immune response that attacked the cancer cells. Another protein, when combined with an antiviral medication (ganciclovir), also killed tumor cells.

The study found this treatment increased survival to about 70%. In addition, any behavioral or cognitive deficits that were caused by the growing tumors disappeared after the tumor was destroyed. The ability to generate an immune response to the cancer cells was also retained by the rat's immune system. Thus, when cancer cells were re-introduced later on, the rat's immune system was able to kill them off on its own.

Obviously, the implications of having a successful treatment for this type of tumor are tremendous, not only for the management of brain cancer, but also for cancer research in general. Phase I clinical trials for the therapy have been scheduled to begin this year. Unfortunately, even if all goes well in clinical trials, this treatment is still years away from being utilized in practice due to the structure of FDA guidelines for drug development. It is, however, a new bright spot on the horizon for gene therapy. It should also be cause for excitement for everyone, gene therapy advocate or not, as it may represent a crucial step toward treating cancer, one disease that has often eluded even our best efforts to do so in the past.