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Developmental Biology - Brain Development

Defects in Frog Brain Can Be Prevented or Repaired

A discovery that bioelectric drugs can prevent or fix brain birth defects in frogs suggests help for humans as well...

A new discovery offers a roadmap for possible therapeutic drugs that could help repair birth defects in humans has been found in frogs. Research led by biologists at Tufts University discovered that brains of developing frog embryos — damaged by nicotine exposure — can be repaired by treatment with certain drugs called "ionoceuticals". Such drugs can drive the recovery of bioelectric patterns in the frog embryonic brain, followed by repair of normal anatomy, gene expression and function in a growing tadpole.

The research, published in Frontiers in Neuroscience, introduces intervention strategies based on restoring the bioelectric "blueprint" for embryonic development, which the researchers suggest could provide a roadmap for the exploration of therapeutic drugs to help repair birth defects.

Earlier studies had shown that nicotine disrupts the normal electrical patterns in the brain of the growing embryo, basically washing out, or reducing the contrast, of the bioelectric blueprint -- a "map" of varying voltage levels around the cells that guides the pattern and growth of tissues and organs. Nicotine in humans has been linked to prenatal morbidity, sudden infant death, attention deficit hypersensitivity disorder (ADHD), and other deficits in cognitive function, learning, and memory, but many questions had remained about how this molecule induces structural defects in the brain.

The authors applied nicotine to developing frog embryos to create neural defects with the intention of identifying specific interventions that could reverse the chemical's harmful effects. Their previous research identified one particular element in the natural electric signalling that controls brain development, hyperpolarization-activated cyclic nucleotide gated channel-2 (HCN2), which was able to restore the bioelectric patterns - much like dialing up the contrast with a photo edit tool - and protect against nicotine-induced defects.

There are two major new discoveries in this study. First, unlike the prior work in which a form of gene therapy modifying the expression of HCN2 was used to repair the defects, the new experiments showed that the same effect can be achieved without introducing a gene - instead, small molecule drugs were used to activate HCN2 channels already present in the frog embryo. Second, the researchers demonstrated that the electrical patterning information that governs brain development can be reset from a distant location on the embryo.
"What was remarkable about the experiments in this study is that when we increased expression of HCN2 at a distance from the brain, in non-neural regions, the defects in the brain were still repaired or prevented. We saw that HCN2 in one part of the embryo could restore the bioelectric pattern not only locally, but at a distance as well."

Michael Levin PhD, Vannevar Bush Professor of Biology, Tufts University, School of Arts and Sciences, Director, the Allen Discovery Center at Tufts.

"The instructions to build a fully grown animal, including organs as complex as the brain, are distributed among all the cells of the embryo," adds Levin. "These results suggest that we might not have to directly target the damaged region, and we can use drugs instead of genetic manipulation, which opens a lot of opportunities for biomedical deployment."
In a developing embryo, bioelectric signals help guide the patterning of tissue and organ formation, as well as regeneration after injury. They are formed by electrically charged ions moving in and out of cells to create voltage differences across the cell membranes.

The pattern of voltage differences among the entire ensemble of cells in the embryo helps guide asymmetry between left and right sides of the body, the formation and development of heart, muscle, limbs and face, and of course the growth and organization of the most complex organ in the body - the brain.

A computational model of an ensemble of cells representing an embryo and its electrical patterns confirmed that rescuing normal brain development from nicotine damage did not require specific targeting of the damaged region. HCN2 increases the hyperpolarization of a cell (increased internal negative charges), so when the model was asked to hyperpolarize a small patch of tissue far from the brain, that patch could propagate and restore polarization of regions all the way to the brain, setting the stage for normal development.
"When thinking about birth defects, especially involving the brain, these results suggest that we don't need to target the specific region that is damaged. We can place the fix almost anywhere in the embryo, and the information will communicate with the rest of the embryo to reset the body's instructions back to normal. That led us to think, could we find a drug that activated HCN2, and use that to prevent defects anywhere in the embryo, or even repair defects that are already underway?"

Vaibhav Pai PhD, Research Scientist, the Allen Discovery Center, Tufts University and first author of the study.

Drugs that activate HCN2 exist - lamotrigine and gabapentin - and are already FDA approved for other indications.

Researchers again exposed frog embryos to nicotine, then treated them with these drugs at different stages of development.

Nicotine-exposed embryos that were not treated with drugs developed with about 68% of brain defects in tadpoles. By comparison, treatment of nicotine exposed embryos with lamotrigine or gabapentin led to significant reduction in brain defects (as few as 10% and 16% of tadpoles, respectively).

Restoration extended beyond electrical and physically observed defects. The authors demonstrated that nicotine-exposed tadpoles treated with the drugs not only restored expression of genetic markers of normal brain development, but remarkably, exhibited normal learning capacity (e.g. training to avoid red light), which was lost in untreated nicotine-exposed tadpoles. This shows a very complete rescue from molecular histology to behavior.

Embryonic exposure to the teratogen nicotine results in brain defects, by disrupting endogenous spatial pre patterns necessary for normal brain size and patterning. Extending prior work in Xenopus laevis that showed that misexpression of ion channels can rescue morphogenesis, we demonstrate and characterize a novel aspect of developmental bioelectricity: channel-dependent repair signals propagate long-range across the embryo. We show that distal HCN2 channel misexpression and distal transplants of HCN2-expressing tissue, non-cell-autonomously reverse profound defects, rescuing brain anatomy, gene expression, and learning. Moreover, such rescue can be induced by small-molecule HCN2 channel activators, even with delayed treatment initiation. We present a simple, versatile computational model of bioelectrical signaling upstream of key patterning genes such as OTX2 and XBF1, which predicts long-range repair induced by ion channel activity, and experimentally validate the predictions of this model. Our results and quantitative model identify a powerful morphogenetic control mechanism that could be targeted by future regenerative medicine exploiting ion channel modulating drugs approved for human use.

Vaibhav P. Pai, Javier Cervera, Salvador Mafe, Valerie Willocq, Emma K. Lederer and Michael Levin.

Vaibhav P. Pai performed experiments. Vaibhav P. Pai and Michael Levin designed the experiments and interpreted the data. Valerie Willocq assisted with the geometric morphometric analysis and in situ hybridization assays. Emma K. Lederer assisted in membrane voltage dye analysis. Javier Cervera and Salvador Mafe designed and performed the simulations. Vaibhav P. Pai and Michael Levin wrote the manuscript together.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics Statement
All experiments were approved by the Tufts University Animal Research Committee (M2017-53) in accordance with the guide for care and use of laboratory animals.

We gratefully acknowledge the support of the Allen Discovery Center program through The Paul G. Allen Frontiers Group (12171), the Templeton World Charity Foundation (TWCF0089/AB55), and the National Institutes of Health (AR055993-01, AR061988), as well as the generous support of Judy Pagliuca. JC and SM acknowledge the support from the Ministerio de Ciencia, Innovación y Universidades (Spain), and the European Regional Development Funds (FEDER), project No. PGC2018-097359-B-I00.

This research was supported by the Paul G. Allen Frontiers Group (12171), G. Harold and Leila Y. Mathers Charitable Foundation (TFU141), the Templeton World Charity Foundation (TWCF0089/AB55), the Ministerio de Ciencia, Innovación y Universidades (Spain) and the European Regional Development Funds (FEDER) (PGC2018-097359-B-I00), and the National Institutes of Health (AR055993-01, AR061988). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

We thank Erin Switzer, and Rakela Colon for Xenopus husbandry and general lab assistance, Dany Adams for help with microscopy, Douglas Blackiston for help with learning behavior assay and analysis, Valerie Schneider for otx2 anti-sense probe, Gerald Eagleson for xbf1 anti-sense probe, Nian-Qing Shi and Bin Ye for Hcn2-WT and Hcn2-DN constructs. We thank Jean-Francois Pare and Joan Lemire for assistance with the cloning of cDNAs into injection vectors PCS2. We thank Kelly McLaughlin and Joshua Finkelstein for their helpful suggestions and comments on the manuscript.

About Tufts University
Tufts University, located on campuses in Boston, Medford/Somerville and Grafton, Massachusetts, and in Talloires, France, is recognized among the premier research universities in the United States. Tufts enjoys a global reputation for academic excellence and for the preparation of students as leaders in a wide range of professions. A growing number of innovative teaching and research initiatives span all Tufts campuses, and collaboration among the faculty and students in the undergraduate, graduate and professional programs across the university's schools is widely encouraged.

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May 27 2020   Fetal Timeline   Maternal Timeline   News

Nicotine induced defects in the frog embryo brain (CENTER) can be rescued by transplanting an HCN2 expressing patch on the embryo far from the brain. Treated embryos are observed to have
normal brain morphology and function (RIGHT). View of normal embryo head is shown at left.
Similar results are seen when nicotine-exposed embryos are treated with ionoceutical drugs.
(FB = forebrain; MB = midbrain; HB = hindbrain) CREDIT Vaibhav Pai, Tufts University

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