Posts Tagged ‘amyotropic lateral sclerosis (ALS)’

Critical Gene in ALS

Larry H Bernstein, MD, FCAP, Curator


Role Found for Critical Gene in 95% of ALS

Like a bad teenager, in 95 percent of all amyotrophic lateral sclerosis (ALS) cases, a protein called TDP-43 leaves its home— the nucleus of motor neuron cells—to congregate, in suspect fashion, in the cytoplasm.In a study published in Science this summer, the Johns Hopkins University (JHU) team of pathologist Phillip Wong, Ph.D., offered new insight into this molecular rebellion. It confirmed a function of normal TDP-43 in the nucleus: orchestrating proper RNA splicing and exon formation. It confirmed what lack of nuclear TDP-43 does: creates improper “cryptic” exons. And it identified proteins that mitigate effects of nuclear TDP-43 loss: potential drug leads.

“The recently published work in Science very clearly demonstrates that in the absence of TDP-43, RNA is misspliced, and in many cases targeted for degradation,” University of Michigan neurologist Sami Barmada, M.D., Ph.D., told Bioscience Technology. Barmada was uninvolved with the research. “A very real consequence of such dysfunctional RNA splicing and degradation is an inability to maintain cell health, ultimately resulting in neuron loss. The authors assembled an intriguing story that tells us quite a bit about how TDP-43 functions, and what happens to those functions in diseases such as ALS, and fronto-temporal dementia (FTD). If the mechanism identified in this manuscript does indeed underlie toxicity due to TDP-43 mislocalization, then it may very well contribute to neuron loss in the vast majority of ALS.”

Medical Research Council (MRC) Laboratory of Molecular Biology neurologist Jernej Ule, Ph.D., toldBioscienceTechnology: “Cryptic exons are interesting. I’m glad that this study will raise the interest in them.” Ule was also uninvolved with the new work.

TDP-43’s central role

For about a decade, it has been known that TDP-43 plays a role in ALS. In 2011 and 2012, a series of papers demonstrated “the profound role of TDP-43 in RNA splicing,” Barmada said. Those papers came out of the labs of University of California San Diego neurologist Don Cleveland, Ph.D.; Ule; University of Toronto neurologist Janice Robertson,Ph.D.; University of Texas Southwestern Medical Center neurologist Gang Yu, Ph.D.; and University of Milan neurologist Antonia Ratti, Ph.D.

Since then, while it has remained unclear “precisely how TDP-43 modulates splicing, we know that it many cases it enhances exon inclusion, while in others it reduces exon inclusion,” Barmada said. In that same 2011 to 2012 time period, “[Washington University neurologist] Youhna Ayala and her colleagues showed that TDP-43 regulates the splicing of its own transcript, targeting it for degradation by RNA decay pathways. So we knew that TDP-43-mediated splicing events can stabilize—or destabilize—RNA.”

The Wong group

For the Science paper, Wong’s group silenced the TDP-43 gene in mouse cells in vitro, then sequenced the RNA. They discovered multitudes of exons were erroneously spliced in: “cryptic exons.” Those cells died.

But when they restored cryptic exon suppression function via other proteins, cells without TDP-43 survived.

The team also silenced TDP-43 in human cells in vitro. Many cryptic exons again appeared in the mRNA.

When the team examined autopsied brain tissue of ALS patients, the same cryptic exons were found in mRNA—but none in controls.

The crew also found evidence of a “positive-feedback loop.” Normal proteins left ungenerated thanks to cryptic exons and TDP-43 depletion included RANBP1. RANBP1 enables the import of TDP-43 into the cell nucleus. The team hypothesized that TDP-43 depletion in the nucleus, leading to less cryptic exon suppression, would result in less functional RANBP1. This may then reduce TDP-43 levels in the nucleus even more.

Suppressing cryptic exons:  not TDP-43’s main role

As noted, Barmada believes the work confirms that, without working TDP-43, RNA is misspliced, resulting in neuron loss. The study did not, however, convince him the main function of normal TDP-43 is to suppress cryptic exons. “What this manuscript tells me is that inappropriate RNA splicing leads to destabilization,” he told Bioscience Technology. “TDP-43, by virtue of its ability to bind thousands of RNA transcripts, is in a unique position to modulate the splicing of a significant fraction of the expressed genome. Abnormal localization or levels of TDP-43 can therefore have dramatic downstream consequences for RNA splicing and subsequent RNA degradation.”

Barmada said, furthermore, he suspects the link to ALS is even more complicated .

“Cytoplasmic TDP-43 mislocalization is a pathologic hallmark of over 95 percent of ALS, and over half of FTD,” he said. “Cytoplasmic TDP-43 deposition is truly lethal for neurons. The study by Wong and colleagues suggests that nuclear TDP-43 depletion mimics the effects seen in FTD, but there also may be a direct and toxic role for excess TDP-43 in the cytoplasm. TDP-43’s RNA binding pattern is different in the cytoplasm, and it forms ribonucleoprotein particles (RNPs) that may sequester RNA and prevent its translation. My guess is that it’s a combination of both: absence of TDP-43 from the nucleus—resulting in aberrant splicing and RNA decay—and excess cytoplasmic TDP-43, sequestering RNA in RNPs.”

Ule told Bioscience Technology he believes it is important to look at cryptic exons.“We’ve been studying them now for a few years, starting with a study of hnRNP [heterogeneous RNP] C-repressed exons a couple of years ago..”

Does TDP-43 stand out?

However,  Ule said, “in the case of TDP-43, I’m still not sure that its role in regulating cryptic exons stands out compared to other hnRNPs. It seems likely to me that most hnRNPs regulate some cryptic exons, though this remains to be fully examined.”

Moreover, said Ule, who (as noted) generated a seminal early paper finding TDP-43 involved in RNA splicing: “TDP-43 regulates many alternative exons as shown by many papers, including one of ours. This study doesn’t convince me that the role of cryptic exons stands out compared to other exons that TDP-43 regulates.”

Finally, he noted, a “huge number of splicing changes occurs in FTD tissues” as shown in his workand that of others “so again, the fact that a few cryptic exons are also misspliced doesn’t convince me that they have a causative role. A more systematic analysis of other model systems would be required for this.”

Mount Sinai Icahn School of Medicine’s Alan Renton, Ph.D., co-wrote a 2014 NatureNeurosciencereview hailing the “landmark” discovery of links between TDP-43, ALS, and FTD. The new paper, he told BioscienceTechnology, “presents good evidence from mouse and human cell culture models that an important normal role of TDP-43 is to suppress the cryptic exon inclusion. The cell model data shows this ability of TDP-43 affects a subset of genes, for some of these genes cryptic exon inclusion appears to neutralize expression from one or both gene copies, and cryptic exon inclusion can sometimes cause cell death.”

The new paper, he continued, “corroborated some of these findings in post-mortem brain tissue obtained from ALS or FTD patients and normal controls. For two genes they detected RNA transcripts that included the predicted cryptic exon in disease cases but not controls. Given that TDP-43 is mislocalized to the cytoplasm of brain cells in the majority of ALS patients, this paper suggests resultant cryptic exon inclusion could be a pathogenic mechanism contributing to the neuronal cell death that characterizes this disease.”

However, he added, “this tentative conclusion would be significantly bolstered by in vivo evidence generated in an animal model system such as mouse.”

“Plenty” to do

Barmada agrees there is “plenty” more to do.

“The first question is what these changes in splicing mean for the proteins encoded by these genes,” he told Bioscience Technology. “Are there changes in protein abundance that reflect the differences in splicing and stability? Following from this, what effect will such changes have on the purported function of these proteins?”

These are some of the downstream questions that “look at the consequences of TDP-43-mediated missplicing on neurodegeneration,” he said. “There are also several upstream questions, concerning how or when TDP-43 is depleted from the nucleus or accumulates in the cytoplasm. One might argue that these are the more clinically relevant questions, since buried in their answers may be ways to prevent such dysfunction from occurring in the first place.”

But he certainly agrees with the authors’ contention in the manuscript that “numerous genetic mutations associated with familial ALS-FTD—VCP, GRN, OPTN, ATXN2, SQSTM1, UBQLN2, PFN1, TBK1, and especially C9ORF72—result in TDP-43 proteinopathy, suggesting a convergent mechanism of neurodegeneration.”

“Absolutely. This is an essential feature of ALS. Nearly 95 percent of all people with ALS demonstrate TDP-43 pathology, whether the disease arises sporadically or if it is inherited. Just about everyone who acquires ALS without a family history of the disease will have TDP-43 pathology, and the majority of those with inherited mutations—including all those listed— will exhibit TDP-43 pathology.”

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