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RESEARCH BRIEFS
Defects in Trafficking Protein Linked to Reduced Brain Size and Mental RetardationA mutation in a single protein-transport gene can cause reduced brain size, severe mental retardation, and death, according to a study now online in Nature Genetics (DOI: 10.1038/ng1276) and appearing in print in the January issue.
The top MRIs show the brain of a six-month-old girl with a mutation in the ARFGEF2 gene. The underdeveloped cerebral cortex does not fill the cranium, as does the healthy adult brain in the bottom MRIs. Arrows on the girl's MRIs indicate nodules of stalled neurons in the enlarged ventricles that could not migrate to the cerebral cortex. (Images courtesy of Renzo Guerrini, University of Pisa, and Bernard Chang, BIDMC) "The idea that trafficking proteins inside the cell have such a huge impact on things outside the cell is very surprising," said Christopher A. Walsh, the principle investigator and Bullard professor of neurology at HMS and BID. Walsh and his lab were looking at the genetic cause of autosomal recessive periventricular heterotopia with microcephaly (ARPHM), a recessive disorder with severe brain malformation. Children with this disease have normal features, but their brain does not grow properly. An MRI of the brain shows the ventricle populated with nodules of underdeveloped neural cells. The disease is usually lethal before age 20. To determine which gene was the cause, Walsh and his lab looked at two Turkish families with a history of the disease. They sequenced several candidate genes and pinpointed mutations in ARFGEF2 as the culprit. The gene codes for the BIG2 protein, which regulates the transport of proteins from the Golgi apparatus to the cell surface. If BIG2 is not working properly, the cell membrane does not have the correct surface proteins displayed. "The surface proteins are like keys and locks," said Volney Sheen, an HMS instructor in neurology and first author on the paper. "If a cell doesn't have the correct key, then it can't interact with the locks on other cells." In a healthy brain, these cell-to-cell protein interactions are what move the neurons from the ventricle to the cerebral cortex, a distance that is over a thousand times the cell's length. In a person with ARPHM, the surface proteins stay in the Golgi apparatus and never make it to the cell surface. Without these proteins, cells cannot make their journey to the cerebral cortex, so they stall in the ventricle, forming ARPHM's characteristic nodules. Walsh says these findings may lead to carrier testing for families with the disease. They also shed light on how cellular functions interact for proper brain development. "People think of traffic proteins as housekeeping proteins that act the same in yeast, bacteria, and us," Walsh said. "This research introduces a new level of specificity in something that people thought of as universal." --Nicole Giese
High-voltage Pulses Open Up Study of Gene FunctionTwo HMS researchers have shown that electroporation, a relatively new technique in mammalian cells for opening reversible pores in the membranes by applying an electrical field, may be used along with other methods including RNAi to deliver genetic material to the cell and facilitate analysis of gene function. This rapid, convenient technique was successful in gain-of-function and loss-of-function studies both in vivo and in vitro in the rodent retina, according to an article now online in the Proceedings of the National Academy of Sciences (DOI: 10.1073/pnas.2235688100) and due to appear in print in early January."Genomics has given us tens of thousands of genes," said senior author Connie Cepko, Howard Hughes investigator and professor of genetics at HMS. "We need a way to assay genes that's faster than the current methods." Cepko and Takahiko Matsuda, a research fellow in genetics, injected plasmid DNA into the subretinal space in newborn rats and mice, and then briefly applied pulses of high voltage to the area. Nearly all the rodent pups appeared healthy after the procedure. When the researchers electroporated a green fluorescent protein expression vector into the pups' eyes, about 80 percent of the rats and half of the mice took up the DNA into their retinal cells. By tracking the protein, Matsuda and Cepko could follow the rodents' retinal development. After three to four weeks, they noted that GFP expression was declining. The researchers used similar methods for gain-of-function and loss-of-function studies. For their gain-of-function analysis, they focused on a transcription factor whose functions in the developing rat retina had been determined using a retroviral vector. When analyzed after 21 days, the cells generated by the electroporated retinal cells exhibited glial phenotypes similar to those in the vector study. To test loss-of-function, the researchers injected DNA-based RNAi vectors that produce double-stranded short, interfering RNAs in mammalian cells. The theory behind RNAi is that expression of any gene can be blocked and its function analyzed. The problem has been getting the genetic material into living cells, and for Cepko and Matsuda, electroporation was one solution. Their target was two transcription factors important in photoreceptor development whose loss-of-function phenotypes were known through studies of knockout mice. They found that the RNAi vectors efficiently and selectively silenced expression of the target genes, which led to abnormal photoreceptor development consistent with that of the knockouts. The authors write that electroporation has several advantages over standard methods to deliver DNA to the rodent retina. The method is rapid, safe, and remarkably efficient. It also is flexible since a variety of DNA constructs can be inserted with less of a limitation on size than that of a viral vector. Finally, multiple constructs can be inserted into a single retinal cell. Although electroporation worked in these investigations, Cepko's lab is not currently using it to develop therapies for retinal disease. "This is more a way to understand development and degeneration at a genetic level, so that future therapies can use it as a foundation," she said. Cepko added that other labs are experimenting with electroporation for therapeutic uses. --Nicole Giese |
