21 November 2011. Like the caretaker of a condemned building shutting off the lights for the last time, DNA methyltransferases shut off motor neuron genes during apoptosis. This is according to a paper in the November 16 Journal of Neuroscience by Lee Martin of the Johns Hopkins University School of Medicine in Baltimore, Maryland. The researchers report that the action of methyltransferases was crucial for apoptosis in cell culture and mice. Their work implies that epigenetic methylation during cell death is not static, but malleable. Martin suggested that treatments modulating methyltransferase activity might temporarily maintain neurons during injury or disease, such as amyotrophic lateral sclerosis (ALS).
Epigenetic tags control processing of DNA without altering its sequence. They tell the transcriptional machinery which genes to turn on and which to switch off. Histone tags, primarily acetylations, regulate many processes, including learning and memory (see ARF related news story on Peleg et al., 2010). They have been linked to schizophrenia (see Schizophrenia Research Forum story) and longevity (see ARF related news story on Greer et al., 2011). DNA methyltransferases (Dnmts) are enzymes that silence genes by adding methyl groups to cytosines in cytosine- and guanine-rich areas known as CpG islands. These islands are common in promoters. DNA methylation has mostly been studied in cancer, but aberrant methylation patterns are known to be associated with Alzheimer’s disease (see ARF related news story on Mastroeni et al., 2009), Rett syndrome (Kondo et al., 2000), and synaptic plasticity (Levenson et al., 2006).
Martin suspected a direct role for DNA methylation in neurodegeneration. He hypothesized that when motor neurons die in ALS, the cell uses methylation to turn off survival factors. Martin recruited graduate student and first author Barry Chestnut—who has since moved on to a postdoc at the National Cancer Institute in Bethesda, Maryland—from a cancer lab to take a look at methylation in ALS.
Using methylation-sensitive restriction enzymes to chop up the genome, Chestnut deduced that DNA methylation patterns in people who had ALS were very different from those in control subjects. To study the link between methylation and motor neuron death, he turned to cultured NSC34 cells—a fusion of spinal cord motor neurons with neuroblastoma cells—. Chestnut focused on Dnmt1, a maintenance enzyme that adds methyl groups to new DNA strands during replication, and Dnmt3a, which catalyzes de-novo methylation when post-mitotic cells turn off genes. He quickly ran into a problem overexpressing Dnmt3a—the cells just started dying. “They were very apoptosis-like,” said Martin, with caspase expression typical of the process. In contrast, overexpressing Dnmt1 did not kill the cells.
How does excess Dnmt3a kill neurons? Surprisingly, Chestnut and colleagues discovered that the methyltransferase tagged with green-fluorescent protein not only turned up in the nucleus, as one would expect, but also in mitochondria and synaptic terminals. Scientists didn’t know previously that Dnmts appeared outside the nucleus in neurons, said Courtney Miller of the Scripps Research Institute, Jupiter, Florida, who was not involved in this work. The question, of course, is what the enzymes could be doing in mitochondria and synapses. Methylating DNA is a possibility that would be a “striking” result, said Bernard Futscher of the University of Arizona in Tucson, noting that the mitochondrial genome is normally unmethylated. Futscher, who was not involved in the study, said he would like to see direct evidence for methyl groups on mitochondrial DNA. NSC34 cells are a cancerous, immortalized line, he added, so their biology with respect to cell death could be abnormal.
Rajiv Ratan of the Burke Medical Research Institute in New York pointed out that histone deacetylases were once thought to only act on histones, but are now known to acetylate cytoplasmic proteins such as tubulin. It is possible that the enzymes are methylating something other than DNA, said Ratan, who was not an author on the study. Another member of the Martin team is now looking into what mitochondrial genes Dnmt might target, Martin said. He also suggested that DNA methylation at this location could be a mechanism of synaptic plasticity.
To study the role of Dnmts in cell death, Chestnut induced apoptosis in NSC34 cells. Within hours, expression of both Dnmt1 and Dnmt3a increased. The researchers then blocked Dnmt activity or expression in apoptotic cells. This was not particularly effective for Dnmt1, but was for Dnmt3a, so the team concluded that Dnmt3a was more important for the cells to complete apoptosis. Methylated cytosine accumulated in the nuclei of apoptotic cells, but this methyl-cytosine disappeared if Dnmt3a was inhibited.
To explore the physiological relevance, the researchers examined a mouse model of motor nerve damage. Chestnut cut the sciatic nerve where it exits the spinal cord, which causes apoptosis in 80 percent of nerves (Martin and Liu, 2002; Martin et al., 2005). Dnmt1, and particularly Dnmt3a, rose in the dying cells less than a week after lesioning. Dnmt3a accumulated in mitochondria as well as in synaptic terminals, and within two days of the lesion, methylated cytosine accumulated in the dendrites, nucleus, and cell body, including mitochondria.
In time, methylation increases and the cells get smaller and smaller, Martin said. That shrinkage was preventable. When the researchers infused the mice with RG108, a general Dnmt inhibitor, it not only prevented cell shrinkage and apoptosis, but the motor neurons grew even larger than normal. “Apparently, motor neurons like to have their genomes hypo-methylated,” Martin said. He suggested this kind of treatment could perhaps sustain motor neurons after axon injury.
Finally, the researchers returned to human ALS tissue. ALS motor neurons contained more Dnmt1 and Dnmt3a than other motor neurons. “It is rather striking,” Martin said. “There is no doubt in my mind that in the ALS motor cortex, motor neuron DNA is getting extensively hyper-methylated.” Drugs like RG108 might help. Dnmt inhibitors are under trial as cancer treatments, noted Jian Feng, at the Mount Sinai School of Medicine in New York. Any anti-methylation treatment would have to be specific for motor neurons to avoid side effects that could result from activating silenced genes across the body, noted Mark Mattson of the National Institute on Aging in Bethesda, Maryland. For example, inhibiting Dnmt can block memory formation, said Yitshak Francis of Columbia University in New York (Miller and Sweatt, 2007). In addition, methyltransferases would probably only block ALS symptoms. It would just be keeping the lights on until doctors fix the root cause of the problem.—Amber Dance.
Chestnut BA, Chang Q, Price A, Lesuisse C, wong M, Martin LJ. Epigenetic regulation of motor neuron cell death through DNA methylation. J Neurosci. 2011 Nov 16; 31(46):16619-16636. Abstract