Editing the brain: How new epigenetic tools could rewrite our understanding of memory and more

During the past decade, it has become clear that epigenetic mechanisms play a particularly active role in the brain. Research by UAB neurobiology chair David Sweatt, Ph.D., for example, proved that it is impossible to form and store new memories without epigenetic tags. Epigenetic dysregulation, other researchers have shown, is involved in many neurological disorders, including Alzheimer's disease, schizophrenia, depression and addiction.

"Just about any neuronal phenomenon can be related to the central epigenetic programming of cell differentiation or cell function or information storage," said Jeremy Day, Ph.D., assistant professor in the UAB Department of Neurobiology, whose lab is investigating the effects of epigenetics in learning, memory and addiction.

Now, an emerging set of molecular tools is giving scientists the ability to manipulate the epigenome in unprecedented ways. In two recent review papers, Day and UAB colleagues capture the excitement surrounding these new "precision epigenetic editing" techniques, which can add and erase epigenetic marks at specific locations throughout the genome.

Researchers are already using the tools to gain a deeper understanding of epigenetic mechanisms in health and disease. They can also contemplate experiments that sound like science fiction, such as creating -- and deleting -- memories. And discoveries made using these tools could pave the way to a "new era of epigenetic therapeutics," as Day writes in the journal Dialogues in Clinical Neurosciences. The epigenetic drugs of the future could reverse aging-related memory impairments and inherited disorders, extinguish the traumatic thoughts of post-traumatic stress disorder, and boost cognitive function.

Control-Alter-Delete

Epigenetic editing strategies typically utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats) or TALE (transcription activator-like effector) systems. Both systems are derived from bacteria and can be programmed to home in on specific genes, carrying an epigenome-modifying enzyme as cargo. To silence a targeted gene, a researcher can deliver an enzyme such as DNA methyltransferase (DNMT). Delivering an enzyme such as histone acetyltransferase, on the other hand, can activate a targeted gene.

TALEs, a slightly older system, require the scientist to create a customized protein to act as the homing device. CRISPRs, which use a more easily generated RNA sequence as a guide (see this graphic from The Scientist magazine), are quickly becoming the tool of choice.

Until now, researchers have been limited to using drugs such as histone deacetylase inhibitors, which "block epigenetic changes throughout the genome instead of targeting one specific gene," Day said. Those globally acting drugs have allowed researchers to establish correlations between an epigenetic change and a behavior or disease, but not prove that one is directly linked with the other. With the new precision-editing tools, investigators can add and remove epigenetic tags one by one. That will allow them to make causal connections, and identify the most important epigenetic modifications in a particular condition. They can then re-create those modifications to verify their findings. "We're going from simply being able to observe changes to being able to manipulate and recapitulate those changes in a controlled way," Day said.

"Let's say you found a modification that is associated with memory," he continued. "Now you can ask, if we generate that change specifically, can we create a memory? Can we implant a memory in the brain by changing epigenetic status? No one has been able to ask those kinds of questions before."

Using next-generation gene sequencing, researchers can also catalog the entire complement of epigenetic changes involved in forming a new memory, for example, or in a specific disease, Day says. "These new tools will allow us to understand which of these changes are most important."

Using optoepigenetic tools, Day can produce gene-specific epigenetic modifications when a light is switched on or off. Each of the waveforms above represents an individual neuron firing an action potential in the brain in real time. The researchers can then correlate the firing properties of these neurons to specific behavioral events when an animal is learning a new task.

Using optoepigenetic tools, Day can produce gene-specific epigenetic modifications when a light is switched on or off. Each of the waveforms above represents an individual neuron firing an action potential in the brain in real time. The researchers can then correlate the firing properties of these neurons to specific behavioral events when an animal is learning a new task.

Making Memories With Light

With another emerging technique, known as optoepigenetics, researchers can also control when an epigenetic change is made. "For certain research questions, you want the manipulation to be temporally precise," Day said. Epigenetic changes associated with memory formation, for instance, may occur in a matter of minutes.

Previous techniques took weeks to produce a given epigenetic change. But researchers can now use an ingenious system developed at MIT that takes advantage of the light-activated protein Cryptochrome 2 (Cry2). Cry2 helps the plant Arabidopsis bend toward sunlight. When it is exposed to light, Cry2 changes its shape and binds to a partner protein, CIB1. Using a TALE as a delivery vehicle, researchers deliver Cry2 to the gene of interest. They then introduce CIB1, fused with an epigenetic-modifying enzyme, into the same region. Finally, they implant a fiber-optic cable connected to a light source into the area. When the light is switched on, CIB1 binds with Cry2, delivering the enzyme, and the desired epigenetic change is made -- in as little as 30 minutes.

Another benefit of optoepigenetics, Day says, is that it can be applied to specific brain regions and cell types, and even isolated to neural projections that connect one brain region to another.

Extinguishing Memories, Reversing Inherited Diseases

The "end game" of all this research, Day says, is to develop new epigenetic therapeutics. In a paper now online in the journal Annual Review of Pharmacology and Toxicology, Day, Sweatt and Andrew Kennedy, Ph.D., a postdoctoral fellow in the Department of Neurobiology, identify four areas that could be the first to benefit from epigenetic therapeutics: PTSD, depression, schizophrenia and cognitive function. In each case, growing evidence from animal and human studies points to epigenetic dysfunction as a factor in disease progression.

For example, epigenetic mechanisms "contribute to the formation and persistence of fear memories" in PTSD, according to one current hypothesis, the authors write. Altering epigenetic marks could allow for "enhanced extinction of conditioned and contextual fear" and could be used in conjunction with cognitive behavioral therapy.

Epigenetic therapies have many other exciting potential applications. They could be used to silence mutated genes that produce damaging proteins, such as the huntingtin protein responsible for Huntington's disease. That same approach, in reverse, could be used to switch on previously silenced genes to treat other conditions. Although we receive copies of genes from both our parents, one of these copies, known as an allele, can be silenced by epigenetic marks during development. In a disease such as Angelman syndrome, which is associated with severe intellectual and developmental disabilities, the active, maternal allele is mutated and nonfunctional, while the paternal allele is silenced. Removing the epigenetic tags from that paternal allele should allow it to start producing the missing protein.

Day's lab is particularly interested in new therapies for addiction. "We think that epigenetic changes resulting from exposure to drugs of abuse may endure for a long time in an addicted individual," Day said. That would explain why an addict who stays clean for several years can be immediately thrown back into a pattern of abuse by exposure to a single trigger, such as a visual cue, he says. "If we can manipulate those changes, it would be a really powerful therapeutic approach."

There are still many challenges to overcome before precision epigenetic therapies reach the clinic. Identifying the correct treatment targets will require a great deal of further basic research, Day says. Investigators will also have to perfect new, human-friendly delivery methods for these therapies; the direct injections and viral vectors used with animal models aren't feasible for clinical use. Nevertheless, "it's a really exciting time," Day said. "We can now start to answer the questions that everyone has been asking, given that epigenetic changes are present in so many conditions."

Editing the Epigenome

New precision techniques allow researchers to add and erase epigenetic marks at specific genes. Because these marks are associated with a growing list of diseases, including depression and Alzheimer's, the new techniques promise to advance understanding and could point to novel treatment options.

Epigenetic "Writers"

Methylation usually silences genes. To methylate a specific gene, researchers link a DNA methyltransferase (DNMT) enzyme to a delivery vehicle such as a transcriptional-activator like effector (TALE) that has been programmed to home in on that gene.

By adding epigenetic marks to histone proteins, researchers can increase gene expression. DNA is wrapped around histones like thread around a spool. Using a TALE to deliver a histone acetyltransferase such as Creb binding protein (CBP), researchers add acetyl groups to the protruding histone tails. This "relaxes" the histones, opening the binding site of the gene of interest to increased transcription by the body's RNA-production machinery.

Epigenetic "Erasers"

Removing methyl groups on DNA tends to boost gene expression. This is a two-step process. First, researchers introduce Tet1, a methylcytosine hydroxylase. Then they target thymine DNA glycosylases (TDG) to the same site to demethylate the gene.

Erasing marks from histones decreases gene expression. Researchers direct a histone deacetylase such as HDAC2 to the gene site, where it removes acetyl groups from histone tails. This blocks the body's RNA-production machinery from accessing the gene.

Optoepigenetics: Using Light to Switch Genes On and Off

Combining the light-sensitive protein cryptochrome 2 (Cry2) with site-specific delivery tools allows researchers to add or erase epigenetic marks in a matter of minutes.

When Cry2 is exposed to blue light, it alters its shape and binds to another protein, CIB1. Scientists take advantage of this conformational change to trigger an epigenetic change.

First, Cry2 is bound to a delivery vehicle, such as a transcriptional-activator like effector (TALE), that has been programmed to home in on a specific gene. At the same time, CIB1 is bound to an epigenetic effector protein, which either adds or erases epigenetic marks, according to the needs of the experiment.

Cry2 waits at the target gene and CIB1 lingers nearby. When researchers are ready to make their epigenetic change, they switch on blue light through an implanted optical fiber. The light triggers Cry2 to alter its shape and bind to CIB1. That delivers the epigenetic effector to the target gene.

Using viral tools, researchers can limit changes to specific brain regions, such as the prefrontal cortex, and even to certain cell types and pathways, such as dopamine neurons that project from the ventral tegmental area to the nucleus accumbens.

The Brave New World of Epigenetic Drugs

Although they are still only in the theoretical stage, drugs that can precisely add or erase epigenetic marks could have several unique advantages over traditional therapies. In a recent paper in the journal Annual Review of Pharmacology and Toxicology, UAB neurobiologists Jeremy Day, Ph.D., Andrew Kennedy, Ph.D., and David Sweatt, Ph.D., explained these potential game-changing attributes.

A "once-in-a-lifetime" pill

Many traditional drugs require continued dosing to maintain a therapeutic effect. But by taking advantage of natural self-perpetuating mechanisms that maintain epigenetic marks, a single dose of an epigenetic drug "could last a lifetime," Day said.

Multigenerational medicine

Although the topic is still a matter of hot debate, there is strong evidence that epigenetic marks can be passed down from parents to children. That means reversing an inherited trait could benefit not just the patient but his or her progeny as well. "This represents an entirely different type of pharmacodynamics," the researchers noted in their paper: "a drug effect in the absence of the organism ever having directly experienced the drug."

Precision-targeted therapy

Epigenetic drugs also have the potential for unparalleled specificity. Traditional drugs generally work by blocking receptors on the cell membrane, Day says. But there may be thousands of these for any particular cell. An epigenetic drug, on the other hand, could silence the gene that codes for that receptor, eliminating it entirely.