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MOUSE BRAIN ATLAS REVEALS NEW CEREBRAL SUBREGIONS, POTENTIAL CELL MARKERS FOR DRUG DEVELOPMENT

ARTICLE IN BRIEF

✓ Neuroscientists reported finding several previously unknown subregions of the hippocampus and cerebellum, as well as what could be new gene markers for major classes of specific cells involved in neurological disorders.

Using genomics and computational neuroanatomy to build a comprehensive three-dimensional atlas of gene expression in the adult mouse, researchers at the nonprofit Allen Institute for Brain Science in Seattle reported several new discoveries about the complexity of the brain and its substructures; this new information could speed the development of new treatments for neurological diseases.

Figure

This image is a cutaway view showing a computationally reconstructed 3D rendering of mouse brain anatomy, with reference planes reflecting a standard 3D coordinate system through the brain.

In the first major paper published since the Allen Brain Atlas was completed in September, neuroscientists reported finding several previously unknown subregions of the hippocampus and cerebellum, as well as what could be new gene markers for major classes of specific cells involved in neurological disorders. The peer-reviewed study appeared online Dec. 6 in an advance publication of the journal Nature.

“This is the first global analysis of simultaneous gene expression and the results are just beginning to come in – this is a scientific foothold,” said co-author and Allen Institute Research Alliance Manager Catherine C. Overly, PhD.

REGIONAL RESTRICTION VARIES

To hone in on the new data, investigators developed an automated process for high-throughput in-situ hybridization in which DNA or RNA interacts so that complexes or hybrids are formed by molecules with similar or complementary sequences. The process detects and locates specific sequences on a specific chromosome.

The atlas has shown that 80 percent of genes are activated in the brain, much higher than the 60 to 70 percent scientists previously believed. In addition, the new findings show that only a small fraction of all genes are expressed at high levels in all cells in the brain, whereas most genes – 75 percent – are each expressed in fewer than 20 percent of brain cells.

Of the 100 genes most specific to each brain region, the team found that expression in some regions, such as the hippocampus and olfactory bulb, gene expression is limited to one area.

The discovery that gene expression is not always restricted regionally may have major implications for understanding and predicting side effects of therapeutic drugs that act through specific proteins, Dr. Overly explained.

NEW MARKERS FOR CEREBRAL CELL TYPES

In addition to identifying all known markers of brain cells, several previously unknown markers of specific cerebral cell types were found. In addition, while the map identified most of the known markers of 12 major brain regions, several genes highly specific for some regions were unknown until now, she told Neurology Today in a telephone interview.

In the hippocampus, which is involved in learning and memory, two previously unrecognized subregions were identified; genes encoding two neural signaling chemicals, the neuropeptides Grp and Nmb, were expressed in the two distinct areas, suggesting functional differences between the subregions, according to Dr. Overly. In addition, expression of the gene Rasgrf1 revealed a previously unknown but large subregion of the cerebellum, which is involved in motor coordination.

Figure

This image shows expression of a specific gene, called etv1, in an area of the brain associated with motivation and reward. The color coding reflects the level of gene expression.

Identification of these subregions is a step toward a more detailed understanding of the working parts of the brain, and may lead to a clearer picture of how the brain executes its functions, she said.

The researchers also searched for genes expressed in each of the major cell types in the brain, finding several previously uncharacterized regulatory genes that appear to be specific to one cell type and govern a cell's unique characteristics and functions.

Identifying new cell-type specific markers may lead to a better understanding of functions in the normal brain as well as diseases in which particular brain cell types are dysfunctional, she noted.

The interactive Web-based tool, the brainchild of Microsoft co-founder Paul G. Allen, who provided $100 million in seed money to launch the project in 2003, presents detailed information on the expression of more than 21,000 genes at the cellular level in the mouse brain. Because humans and mice share about 90 percent of their genes in common, the atlas offers researchers a free opportunity to better understand such diseases as Alzheimer disease, Parkinson disease, epilepsy, schizophrenia, autism, and addiction.

REGIONAL RESTRICTION VARIES

To hone in on the new data, investigators developed an automated process for high-throughput in-situ hybridization in which DNA or RNA interacts so that complexes or hybrids are formed by molecules with similar or complementary sequences. The process detects and locates specific sequences on a specific chromosome.

The atlas has shown that 80 percent of genes are activated in the brain, much higher than the 60 to 70 percent scientists previously believed. In addition, the new findings show that only a small fraction of all genes are expressed at high levels in all cells in the brain, whereas most genes – 75 percent – are each expressed in fewer than 20 percent of brain cells.

Of the 100 genes most specific to each brain region, the team found that expression in some regions, such as the hypothalamus and midbrain, overlaps with expression in other areas. In other regions such as the hippocampus and olfactory bulb, gene expression is far more restricted.

Figure

This shows the expression of a gene called Calb2 in the cortex, the outer layers of the brain thought to be responsible for the most sophisticated brain functions. The color coding reflects the level of gene expression.

The discovery that gene expression is not always restricted regionally may have major implications for understanding and predicting side effects of therapeutic drugs that act through specific proteins, Dr. Overly explained.

Ideally, a drug targets a protein whose associated gene is expressed only in the regions affected by disease. Because drug activity in regions unaffected by disease can cause unwanted side effects, the ability to map expression patterns throughout the brain should help scientists develop drug targets with fewer side effects, she said.

The institute next plans to develop a model of the neocortex and leverage the existing data set for information about autism, ALS, spinal cord problems, epilepsy, schizophrenia, and neurodevelopment. The researchers are also talking to scientists working on other human brain atlas projects and databases in an effort to link them, Dr. Overly said.

EXPERTS COMMENT

“This is wonderful research,” commented Charles D. Stiles, PhD, professor in the department of microbiology and molecular genetics and chair of the neuro-oncology department at the Dana Farber Cancer Institute.

In 2004, Dr. Stiles and other researchers published the first atlas showing the locations of crucial gene regulators in the brain called transcription factors that, when altered, can cause the genes they control to go awry, causing abnormalities in the development or function of nerves and related structures (Science 2004;306:2255–2257).

“When I was first doing research, we believed that about one-third of genes were organ-specific, including those expressed in the brain,” Dr. Stiles said. “As it turns out the brain isn't all that special in that respect.”

The new paper is “comprehensive” and “includes nuggets everywhere, he continued, including some that parallel his research involving multiple transcription factor expression in single cell layers and other “intricate” regional findings. “Genes specific to one brain region or cell type should allow development of transgenic mouse models for neurological disease research,” he noted.

Michael A. Arbib, PhD, professor of biological sciences, biomedical engineering, electrical engineering, neuroscience and psychology at the University of Southern California (USC), noted that the results should help researchers fine tune their study of gene expression and brain development in higher primates.

“It's an exciting study and a fantastic step forward,” he told Neurology Today in a telephone interview.

As Director of the USC Brain Project, Dr. Arbib oversees the integration of research in the neuroscience of synaptic plasticity and visuomotor coordination with neuroinformatics, applying computational techniques to the analysis of the relationship between structure and function in models of interacting brain regions in rats and primates.

Although the data might help target future research to see if genes are expressed in comparable parts of the human brain, his work in synaptic processes and neuroplasticity requires, at the very least, a data set based on primate models, he said.

“For our work, the mouse is too far away. For us the value of the data is limited. What we need is information on the big picture. It can tell us which genes are active in areas that were weren't aware of before and pinpoint specific genes for future studies. This is a learning process and the findings are going to trigger a lot of new research. This is a collaborative effort in every way, and occasionally something clicks.”

TECHNIQUE BEHIND THE ATLAS

The atlas was developed using a technique called “automated high throughput in-situ hybridization” in which microscopically thin slices of brain tissue are taken from different parts of the brain and chemically labelled with messenger RNA probes that bind to individual gene sequences. In-situ maps were made for every gene in the mouse genome, then loaded into an enormous database, linking the data to roughly 85 million images. Using special software available at the Allen Brain Atlas Web site, researchers can compare and navigate image data and the extensive gene expression database, examining gene expression at the cellular level. The Allen Brain Atlas is publicly available for free at www.brain-map.org

REFERENCE

• Lein ES, et al. Genome-wide atlas of gene expression in the adult mouse brain, Nature 2006; E-pub 6 Dec. 2006.