San Diego: TDP-43 Targets Loom Large—But Where’s the Bull’s Eye?

TDP-43, a protein implicated in both frontotemporal lobar degeneration and amyotrophic lateral sclerosis, as well as Alzheimer’s disease (Brouwers et al., 2010), is an RNA-binding protein with an undefined role. If posters at the Society for Neuroscience (SfN) meeting and a recent publication are any indication, the field is poised to discover a wealth of clues to TDP-43 function (see also ARF related news story). Scientists are scanning the transcriptome for TDP-43 target RNAs, and early reports suggest there are quite a lot of them. A paper in the November 4 Journal of Biological Chemistry online describes more than 4,000 RNAs that interact with TDP-43 in cortical neurons, and at the SfN meeting, held November 13-17 in San Diego, California, several poster presentations described TDP-43 targets in cell lines. Scientists hope that they might link a small subset of TDP-43-binding RNAs to pathology. More specifically, altered gene expression might someday provide biomarkers for early diagnosis of ALS, suggested Shangxi Xiao of the University of Toronto, Canada, an author on one of the posters.

What RNAs does TDP-43 modify, how does it change them, and what proteins does it work with? These are questions everybody is asking, said Chantelle Sephton, first author on the JBC paper with senior author Gang Yu and colleagues at the University of Texas Southwestern Medical Center in Dallas. All lead up to the ultimate questions, she said: How do these processes cause pathology, and how might doctors target TDP-43 to treat disease? Therein lies the rub, since TDP-43 has so many binding partners.

I do not believe every target plays a role in the disease process, said Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, who was not involved in the JBC paper or SfN posters. Indeed, transcriptome-scanning studies are merely hypothesis-generating tools, said Joachim Herz, another professor at Southwestern Medical Center and a coauthor on Sephton’s paper.

Foundational Studies
The JBC paper is the first published study to seek out TDP-43 targets without overexpressing the protein, the authors said. The authors isolated RNA from rat primary neuron cultures, then used TDP-43 antibodies to immunoprecipitate RNAs that interact with the protein. Collaborating with researchers in the group of Melissa Moore at the University of Massachusetts, the scientists sequenced the TDP-43 targets. I was floored by the number of targets, Sephton said. More than 4,000 genes appear to rely on TDP-43 to modulate their expression. It’s really a foundation-type study, Sephton said, noting that researchers can now pick and choose which targets they think are involved in disease for follow-up studies.

TDP-43 is bound to sequences of UG repeats in target RNAs, often with a UA in the middle, but Sephton said there may be other TDP-43 binding motifs, too. Using a standard gene ontology database, the researchers identified genetic categories that were enriched in their RNA sample. Genes involved in RNA processing, synapse function, and development were among the top hits.

Among the gene set was TDP-43 itself, suggesting it regulates its own expression via some sort of feedback loop. The authors suggest TDP-43 binds its own 3′-UTR, and alters either the stability or translational efficiency of the RNA. The exact mechanism for this feedback is unclear, Sephton said, but it dovetails nicely with animal studies. Animals heterozygous for TDP-43 often produce more than half the normal amount of the protein, suggesting the remaining allele compensates for the missing one (Sephton et al., 2010). Other TDP-43 targets readers may recognize include FUS, progranulin, tau, amyloid precursor protein, and α-synuclein, which are all related to neurodegeneration.

The researchers also identified proteins that interact with TDP-43. In collaboration with the laboratory of Junmin Peng at Emory University in Atlanta, Georgia, they used mass spectrometry to analyze proteins that co-immunoprecipitated with TDP-43 from rat brain nuclear extracts. They discovered 25 proteins that seem to buddy up with TDP-43 in the nucleus. Most were RNA-binding proteins such as splicing or translation factors, confirming previous results (Freibaum et al., 2010).

Cell Line Studies
Sephton and collaborators, as well as Clotilde Lagier-Tourenne and colleagues at the University of California in San Diego (see ARF related news story), are using brain tissue to investigate TDP-43 targets, but other scientists have started similar studies with cell lines. At the SfN meeting, Shangxi Xiao and Janice Robertson of the University of Toronto, Canada, presented work with TDP-43/RNA co-immunoprecipitation from the SH-SY5Y human neuroblastoma line. The scientists identified 102 TDP-43 targets, with UG repeat and pyrimidine-rich TDP-43 binding sequences. The majority of TDP-43 binding sites were within introns; 23 percent were outside introns. Using RT-PCR, Xiao showed that five out of 50 target genes were downregulated, or had altered splicing patterns, in the spinal cords of people who died of ALS compared to healthy control tissue.

Atsushi Shiga from Niigata University in Japan presented a poster on the gene expression and splicing changes in TDP-43-depleted cells. The researchers used short interfering RNA to dampen TDP-43 expression in HeLa human cervical cancer cells. They found that 123 genes, including several involved in inflammation, were up- or downregulated. Inflammation is a known factor in ALS as well as other neurodegenerative diseases (see ARF related news story). In addition, 892 genes were abnormally spliced in the absence of TDP-43. Many of the latter are involved in the Golgi and other endomembrane systems.

TDP-43, of course, is not the only RNA-binding protein implicated in ALS and FTLD. In a poster, Shinsuke Ishigaki and Gen Sobue reported on early studies knocking down FUS in the spinal cord neuroblastoma hybridoma NSC34. They have identified several FUS targets, Ishigaki told ARF, and hope to perform further studies with primary motor neuron cultures and knockout mice.

The multitude of TDP-43 targets indicates the complexity researchers face in untangling disease mechanisms Gao said. All these toxic proteins affect so many pathways, he told ARF. It is a challenge for the fieldto identify the key targets.

Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han YH, Dewey CM, Roth FP, Herz J, Peng J, Moore MJ, Yu G. Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes. J Biol Chem. 2010 Nov 4. Abstract

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