What goes wrong in the brain of a person with a hexanucleotide expansion in C9ORF72, a protein involved in RNA processing? Quite a bit, according to an analysis published in the July 20 Nature Neuroscience. Researchers from the Mayo Clinic profiled the brain transcriptomes of both C9ORF72-linked and sporadic cases of amyotrophic lateral sclerosis, and discovered that the expansion-toting brains contained many more structurally morphed RNAs, particularly for genes involved in the unfolded protein response and protein trafficking. “It suggests that the mechanisms leading to C9ORF72 and sporadic disease might be very different,” said Mercedes Prudencio of the Jacksonville, Florida, Mayo branch, one of the first authors on the study.
The researchers, led by joint senior authors Leonard Petrucelli in Florida and Hu Li of the Rochester, Minnesota, clinic, have put their data—encompassing RNA expression levels, alternative splicing, and alternative 3’ UTR lengths—online. Other scientists can now compare animal or iPS model changes to human brain regulation. “The public availability of these types of data sets are essential to push the field forward and will be very valuable for testing new hypotheses,” commented Adrian Isaacs of University College London, who was not involved in the study. “We will certainly be mining the data.”
Prudencio and co-first author Veronique Belzil in Florida, who designed the study, wanted to distinguish RNA processing defects caused by mutations in C9ORF72 from those caused by TDP-43, another ALS protein that warps the transcriptome. Avoiding tissues with extensive TDP-43 pathology, such as the spinal cord, they focused on the frontal cortex and cerebellum of eight C9ORF72 cases, 10 sporadic ALS cases with no known mutation, and nine neurologically healthy controls. Though C9ORF72 expansions can also cause frontotemporal dementia, Petrucelli and colleagues stuck to pure ALS in this study.
Along with joint first authors Christian Ross in Rochester and Ranjan Batra, now at the University of California in San Diego, the researchers identified the genes upregulated or downregulated in both kinds of ALS, as well as those alternatively spliced. In addition, they analyzed the presence of alternative polyadenylation sites in the pre-mRNAs. These loci indicate where RNA polymerase should stop copying DNA and add a poly-adenine tail. Alternative sites can lead to longer or shorter 3’ UTRs, which may then harbor different sites for microRNA and regulatory proteins, and alter that RNA’s regulation, Prudencio explained.
The authors hoped to find similarities between C9ORF72 and sporadic cases that would point to common mechanisms or even biomarkers. In that, they were surprised. “We identified two completely different brain profiles,” Belzil said. In C9ORF72 cases, 361 genes were differentially expressed in the cerebellum, and 241 in the frontal cortex. Expression of only 136 genes was altered in each of those regions in sporadic cases. Fifty-seven up- or downregulated cerebellum genes, and 32 in the frontal cortex, were common to both kinds of ALS.
In terms of RNA structure, alternative polyadenylation arose in 1,437 of C9ORF72 cerebellum RNAs and 716 in sporadic cerebella, and in 968 of frontal cortex RNAs of C9ORF72 cases versus 1,266 in sporadic. Alternative splicing occurred in 8,224 genes in the cerebella of C9ORF72 ALS brains, versus 2,229 in sporadic samples. In the frontal cortex, C9ORF72 brains exhibited 902 splicing changes, to 282 in sporadic cases. “It seems C9ORF72 mutations lead to worse RNA defects,” Belzil said.
The authors applied different bioinformatics tools to group the altered RNAs by function. For C9ORF72, misregulation of the unfolded protein response and protein and vesicle transport, including across the nuclear envelope, were particularly common (see genes with altered exon splicing, specifically, in image above). In contrast, alterations in sporadic cases clustered in cellular defenses and synaptic transmission. These differences suggest that treatments, and clinical trials, may need to focus specifically on one or the other ALS type, Prudencio concluded.
She and her colleagues were also surprised by the extent of RNA changes in the cerebellum. The authors wrote that despite the cerebellum’s importance for movement, scientists seem to have overlooked its involvement in ALS. However, their findings are in line with recent studies reporting cerebellar atrophy in the disease (Tan et al., 2014; Irwin et al., 2013). The cerebellum expresses more C9ORF72 than the cortex, as well as more of the C9ORF72 RNA foci and its translated repeat dipeptides, commented Edward Lee of the University of Pennsylvania Perelman School of Medicine in Philadelphia, who was not involved in the paper.
Some of the other gene networks highlighted in the study also match findings from other research models, noted Ana Jovicic of Stanford University in Palo Alto, California, who did not participate in the work. For example, ribosomal RNA (rRNA) processing genes were misregulated in both the cerebella and frontal cortices of C9ORF72 cases, and another lab recently reported crippled rRNA maturation in the blood cells and motor cortices of people who had C9ORF72-based ALS (see Haeusler et al., 2014). The dipeptides created by C9ORF72 translation also have the potential to interfere with rRNA processing (see Aug 2014 news). In addition, Petrucelli and colleagues identified misregulation of nuclear transport genes, a topic that has been getting attention in recent ALS meetings, Jovicic said.
Researchers should be able to make similar comparisons with the new database, Lee said. For example, scientists working with iPS cells in culture might zero in on a new mechanism for disease, but want to check its relevance to human ALS in the brain. “A lot of things could be checked with this data set,” he said.
Plus, this publication is just a first taste, Belzil said. The lab plans to generate a full profile of sporadic and C9ORF72 ALS brains, including epigenetic markers, in the future. They also plan to investigate the transcriptomes of blood samples, still hoping for a good biomarker, and of frontotemporal dementia cases. The group might also follow up on some of the specific genes identified in the transcriptome analysis, Prudencio said, but have not settled on any favorites yet.
In addition, researchers still have to explain how C9ORF72 creates such transcriptional havoc. Prudencio noted that the abnormal RNA foci transcribed from the repeat sequence are known partners of RNA-binding proteins like hnRNPH, and may sequester them from their normal functions (see Cooper-Knock et al., 2014; Lee et al., 2013). Supporting that theory, she and her co-authors noticed that many of the alternatively spliced genes contained an hnRNPH-binding motif.—Amber Dance
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