Tag Archives: disorder

Penn Vet-Temple team characterizes genetic mutations linked to a form of blindness

Achromatopsia is a rare, inherited vision disorder that affects the eye’s cone cells, resulting in problems with daytime vision, clarity and color perception. It often strikes people early in life, and currently there is no cure for the condition.

One of the most promising avenues for developing a cure, however, is through gene therapy, and to create those therapies requires animal models of disease that closely replicate the human condition.

In a new study, a collaboration between University of Pennsylvania and Temple University scientists has identified two naturally occurring genetic mutations in dogs that result in achromatopsia. Having identified the mutations responsible, they used structural modeling and molecular dynamics on the Titan supercomputer at Oak Ridge National Laboratory and the Stampede supercomputer at the Texas Advanced Computing Center to simulate how the mutations would impact the resulting protein, showing that the mutations destabilized a molecular channel essential to light signal transduction.

The findings provide new insights into the molecular cause of this form of blindness and also present new opportunities for conducting preclinical assessments of curative gene therapy for achromatopsia in both dogs and humans.

“Our work in the dogs, in vitro and in silico shows us the consequences of these mutations in disrupting the function of these crucial channels,” said Karina Guziewicz, senior author on the study and a senior research investigator at Penn’s School of Veterinary Medicine. “Everything we found suggests that gene therapy will be the best approach to treating this disease, and we are looking forward to taking that next step.”

The study was published in the journal PLOS ONE and coauthored by Penn Vet’s Emily V. Dutrow and Temple’s Naoto Tanaka. Additional coauthors from Penn Vet included Gustavo D. Aguirre, Keiko Miyadera, Shelby L. Reinstein, William R. Crumley and Margret L. Casal. Temple’s team, all from the College of Science and Technology, included Lucie Delemotte, Christopher M. MacDermaid, Michael L. Klein and Jacqueline C. Tanaka. Christopher J. Dixon of Veterinary Vision in the United Kingdom also contributed.

The research began with a German shepherd that was brought to Penn Vet’s Ryan Hospital. The owners were worried about its vision.

“This dog displayed a classical loss of cone vision; it could not see well in daylight but had no problem in dim light conditions,” said Aguirre, professor of medical genetics and ophthalmology at Penn Vet.

The Penn Vet researchers wanted to identify the genetic cause, but the dog had none of the “usual suspects,” the known gene mutations responsible for achromatopsia in dogs. To find the new mutation, the scientists looked at five key genes that play a role in phototransduction, or the process by which light signals are transmitted through the eye to the brain.

They found what they were looking for on the CNGA3 gene, which encodes a cyclic nucleotide channel and plays a key role in transducing visual signals. The change was a “missense” mutation, meaning that the mutation results in the production of a different amino acid. Meanwhile, they heard from colleague Dixon that he had examined Labrador retrievers with similar symptoms. When the Penn team performed the same genetic analysis, they found a different mutation on the same part of the same gene where the shepherd’s mutation was found. Neither mutation had ever been characterized previously in dogs.

“The next step was to take this further and look at the consequences of these particular mutations,” Guziewicz said.

The group had the advantage of using the Titan and Stampede supercomputers, which can simulate models of the atomic structure of proteins and thereby elucidate how the protein might function. That work revealed that both mutations disrupted the function of the channel, making it unstable.

“The computational approach allows us to model, right down to the atomic level, how small changes in protein sequence can have a major impact on signaling,” said MacDermaid, assistant professor of research at Temple’s Institute for Computational Molecular Science. “We can then use these insights to help us understand and refine our experimental and clinical work.”

The Temple researchers recreated these mutated channels and showed that one resulted in a loss of channel function. Further in vitro experiments showed that the second mutation caused the channels to be routed improperly within the cell.

Penn Vet researchers have had success in treating various forms of blindness in dogs with gene therapy, setting the stage to treat human blindness. In human achromatopsia, nearly 100 different mutations have been identified in the CNGA3 gene, including the very same one identified in the German shepherd in this study.

The results, therefore, lay the groundwork for designing gene therapy constructs that can target this form of blindness with the same approach.

The study was supported by the Foundation Fighting Blindness, the National Eye Institute, the National Science Foundation, the European Union Seventh Framework Program, Hope for Vision, the Macula Vision Research Foundation and the Van Sloun Fund for Canine Genetic Research.

Source: University of Pennsylvania 

Neuroscientists reverse memories’ emotional associations

MIT study also identifies the brain circuit that links feelings to memories.

By Anne Trafton

Most memories have some kind of emotion associated with them: Recalling the week you just spent at the beach probably makes you feel happy, while reflecting on being bullied provokes more negative feelings.

A new study from MIT neuroscientists reveals the brain circuit that controls how memories become linked with positive or negative emotions. Furthermore, the researchers found that they could reverse the emotional association of specific memories by manipulating brain cells with optogenetics — a technique that uses light to control neuron activity.

The findings, described in the Aug. 27 issue of Nature, demonstrated that a neuronal circuit connecting the hippocampus and the amygdala plays a critical role in associating emotion with memory. This circuit could offer a target for new drugs to help treat conditions such as post-traumatic stress disorder, the researchers say.

“In the future, one may be able to develop methods that help people to remember positive memories more strongly than negative ones,” says Susumu Tonegawa, the Picower Professor of Biology and Neuroscience, director of the RIKEN-MIT Center for Neural Circuit Genetics at MIT’s Picower Institute for Learning and Memory, and senior author of the paper.

 This image depicts the injection sites and the expression of the viral constructs in the two areas of the brain studied: the Dentate Gyrus of the hippocampus (middle) and the Basolateral Amygdala (bottom corners). Credits Image courtesy of the researchers/MIT

This image depicts the injection sites and the expression of the viral constructs in the two areas of the brain studied: the Dentate Gyrus of the hippocampus (middle) and the Basolateral Amygdala (bottom corners).
Credits: Image courtesy of the researchers/MIT

The paper’s lead authors are Roger Redondo, a Howard Hughes Medical Institute postdoc at MIT, and Joshua Kim, a graduate student in MIT’s Department of Biology.

Shifting memories

Memories are made of many elements, which are stored in different parts of the brain. A memory’s context, including information about the location where the event took place, is stored in cells of the hippocampus, while emotions linked to that memory are found in the amygdala.

Previous research has shown that many aspects of memory, including emotional associations, are malleable. Psychotherapists have taken advantage of this to help patients suffering from depression and post-traumatic stress disorder, but the neural circuitry underlying such malleability is not known.

In this study, the researchers set out to explore that malleability with an experimental technique they recently devised that allows them to tag neurons that encode a specific memory, or engram. To achieve this, they label hippocampal cells that are turned on during memory formation with a light-sensitive protein called channelrhodopsin. From that point on, any time those cells are activated with light, the mice recall the memory encoded by that group of cells.

Last year, Tonegawa’s lab used this technique to implant, or “incept,” false memories in miceby reactivating engrams while the mice were undergoing a different experience. In the new study, the researchers wanted to investigate how the context of a memory becomes linked to a particular emotion. First, they used their engram-labeling protocol to tag neurons associated with either a rewarding experience (for male mice, socializing with a female mouse) or an unpleasant experience (a mild electrical shock). In this first set of experiments, the researchers labeled memory cells in a part of the hippocampus called the dentate gyrus.

Two days later, the mice were placed into a large rectangular arena. For three minutes, the researchers recorded which half of the arena the mice naturally preferred. Then, for mice that had received the fear conditioning, the researchers stimulated the labeled cells in the dentate gyrus with light whenever the mice went into the preferred side. The mice soon began avoiding that area, showing that the reactivation of the fear memory had been successful.

The reward memory could also be reactivated: For mice that were reward-conditioned, the researchers stimulated them with light whenever they went into the less-preferred side, and they soon began to spend more time there, recalling the pleasant memory.

A couple of days later, the researchers tried to reverse the mice’s emotional responses. For male mice that had originally received the fear conditioning, they activated the memory cells involved in the fear memory with light for 12 minutes while the mice spent time with female mice. For mice that had initially received the reward conditioning, memory cells were activated while they received mild electric shocks.

Next, the researchers again put the mice in the large two-zone arena. This time, the mice that had originally been conditioned with fear and had avoided the side of the chamber where their hippocampal cells were activated by the laser now began to spend more time in that side when their hippocampal cells were activated, showing that a pleasant association had replaced the fearful one. This reversal also took place in mice that went from reward to fear conditioning.

Altered connections

The researchers then performed the same set of experiments but labeled memory cells in the basolateral amygdala, a region involved in processing emotions. This time, they could not induce a switch by reactivating those cells — the mice continued to behave as they had been conditioned when the memory cells were first labeled.

This suggests that emotional associations, also called valences, are encoded somewhere in the neural circuitry that connects the dentate gyrus to the amygdala, the researchers say. A fearful experience strengthens the connections between the hippocampal engram and fear-encoding cells in the amygdala, but that connection can be weakened later on as new connections are formed between the hippocampus and amygdala cells that encode positive associations.

“That plasticity of the connection between the hippocampus and the amygdala plays a crucial role in the switching of the valence of the memory,” Tonegawa says.

These results indicate that while dentate gyrus cells are neutral with respect to emotion, individual amygdala cells are precommitted to encode fear or reward memory. The researchers are now trying to discover molecular signatures of these two types of amygdala cells. They are also investigating whether reactivating pleasant memories has any effect on depression, in hopes of identifying new targets for drugs to treat depression and post-traumatic stress disorder.

David Anderson, a professor of biology at the California Institute of Technology, says the study makes an important contribution to neuroscientists’ fundamental understanding of the brain and also has potential implications for treating mental illness.

“This is a tour de force of modern molecular-biology-based methods for analyzing processes, such as learning and memory, at the neural-circuitry level. It’s one of the most sophisticated studies of this type that I’ve seen,” he says.

The research was funded by the RIKEN Brain Science Institute, Howard Hughes Medical Institute, and the JPB Foundation.