In amyotrophic lateral sclerosis and frontotemporal dementia, loss of the RNA-binding protein TDP-43 from the nucleus creates a surge of mis-spliced mRNAs in neurons. So far, only one of these errant transcripts, stathmin-2, has been tied to disease pathology. Now, two preprints uploaded to bioRXiv on April 4 detail another—UNC13A. Variants in this gene increase risk for ALS/FTD.
- Neurons lacking nuclear TDP-43 mis-splice UNC13A, make less of the protein.
- UNC13A risk variants incorporate cryptic exons.
- This only occurs in brain and spinal cord tissue harboring TDP-43 deposits.
One paper was penned by researchers led by Pietro Fratta, University College London, and Michael Ward, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland. The second is from the labs of Aaron Gitler, Stanford University, California, and Leonard Petrucelli, Mayo Clinic, Jacksonville, Florida. Both papers report that if TDP-43 binding to UNC13A falters, the transcript is mis-spliced and less Unc13A protein is made. In frontal cortex neurons from people who had ALS/FTD with TDP-43 deposits, mis-splicing was highest in cells carrying the UNC13A risk variants. The studies support the idea that sequestering TDP-43 in the cytosol may unleash aberrant splicing events that contribute to neurodegenerative disease pathology.
Huda Zoghbi, Baylor College of Medicine, Houston, praised the thorough experiments from both groups. “They’ve linked two phenomena in ALS—TDP-43 accumulation and risk variants—together with a biological explanation,” she told Alzforum.
In almost all people with ALS and half of those with FTD, TDP-43 gets trapped in the cytosol, where it forms deposits (Sep 2020 news). As TDP-43 protein levels fall in the nucleus, splicing errors that it would normally prevent begin to magnify (reviewed by Klim et al., 2021; Mar 2011 news). One such snafu is the “cryptic exon.” This happens when a piece of an intron mistakenly incorporates into the mRNA, which can lead to cell dysfunction and death. In ALS, cryptic exons can run amok, leading researchers to search for those that might contribute to disease pathology (Aug 2015 news).
Previously, scientists found that depleting TDP-43 from the nucleus prompts a cryptic exon to form in microtubule-binding protein stathmin-2, cutting its protein levels in half (Dec 2018 conference news; Jan 2019 news). The STMN2 cryptic exon occurred only in motor neurons from people with ALS, who had less stathmin-2 protein in their spinal cord tissue. What other errant transcripts might exacerbate ALS/FTD pathology?
To find out, both groups searched for splicing changes caused by the absence of TDP-43. From the Ward and Fratta labs, co-first authors Anna-Leigh Brown, Oscar Wilkins, and Matthew Keuss, all at UCL, and Sarah Hill at NINDS, used CRISPR to stifle TDP-43 expression in human induced pluripotent stem cell (iPSC)-derived neurons, then sequenced the RNA. They found 179 cryptic exons.
Co-first authors Rosa Ma in Gitler’s lab and Mercedes Prudencio and Yuka Koike in Petrucelli’s lab took a different approach to assess splicing variation. They reanalyzed RNA sequencing data from frontal cortex neurons in postmortem brain samples taken from seven older adults who had had FTD or FTD/ALS (Liu et al., 2019). They found 65 differently spliced transcripts in nuclei lacking TDP-43 that they deemed worth of further study.
Splicing Differences. In human iPSC-derived neurons, knocking down TDP-43 (right) upped the percentage of transcripts containing cryptic exons, including those in UNC13A and stathmin-2 (STMN2). Each circle denotes a splice junction. [Courtesy of Brown et al., bioRXiv, 2021.]
The researchers were interested in UNC13A because of its association with ALS and FTD (Mar 2018 news; Apr 2018 news). Named for the uncoordinated movements of C. elegans when the gene is mutated, UNC13A is a member of a family of proteins that regulate axon function and neurotransmitter release. In mice, deleting UNC13A causes motor neuron defects (Sept 2009 news).
New Cryptic Exon. Compared to UNC13A splicing in control iPSC-derived neurons (teal), the transcript in cells with TDP-43 knocked down (gold) harbored a cryptic exon. Single-nucleotide polymorphisms (SNPs) in the intron and cryptic exon region (CE) close to the TDP-43 binding site (green square) made splicing even worse. [Courtesy of Brown et al., bioRxiv, 2021.]
Lo and behold, both groups identified the same cryptic exon between exons 20 and 21 (see image above). Like the STMN2 cryptic exon, the UNC13A one contains a stop codon, which the authors believe causes nonsense mediated decay of the mRNA. After knocking down TDP-43 in SH-SY5Y cells and iPSC-derived neurons, the cryptic exon appeared, while normal Unc13A mRNA and protein waned.
What about in people? Brown and colleagues analyzed the same human RNA-Seq dataset as Ma and colleagues did, finding cryptic exons only in nuclei devoid of TDP-43. Within medial frontal lobe neurons from four people with FTD, Ma and colleagues likewise saw the UNC13A cryptic exon only in nuclei lacking TDP-43 (see image below). “UNC13A risk alleles might act as an Achilles’ heel—lurking under the surface, not causing problems until TDP-43 becomes dysfunctional,” Ma and colleagues wrote in their preprint.
Looking more broadly, both groups sequenced RNA from brain and spinal cord tissue extracts taken from people with ALS or FTD and controls in the New York Genome Center (NYGC) ALS Consortium. They found the cryptic exon only in people who had accumulated TDP-43 deposits. Brown and colleagues detected the transcript in 89 percent of people who had FTD and 38 percent of those who had ALS. Ma and colleagues saw it in almost half of FTD cases. They also found it in 63 frontal cortex tissue samples from 49 FTD cases or healthy controls from the Mayo Clinic Brain Bank. In the NYGC samples, cryptic exon expression mirrored TDP-43 deposits, appearing in the spinal cords and motor cortices of ALS cases and frontal and temporal cortices in FTD cases.
Where Risk SNPs Come In
In the UNC13A gene, two single-nucleotide polymorphisms (SNPs) are known that increase a person's risk of ALS/FTD: rs12973192 in the cryptic exon and rs12608932 in the nearby TDP-43 binding region. Both research groups found that these variants weakened TDP-43 binding to UNC13A. Brown and colleagues believe this decreased binding promoted cryptic splicing.
How do these alleles increase disease risk? Looking through whole-genome sequencing (WGS) data from the NYGC cohort, Brown and colleagues found that people who were homozygous for either SNP had more UNC13A cryptic exon transcripts than did heterozygotes or noncarriers. The amount of UNC13A transcripts with the cryptic exon rose with that of STMN2 cryptic exons, which they previously found to correlate with TDP-43 deposits (Prudencio et al., 2020; commentary by Glass 2020). Ma and colleagues found that people with FTD/ALS who carry the rs12973192 risk SNP expressed the cryptic exon in almost every UNC13A transcript, while those with the common allele had up to three times less.
Understanding how loss of the UNC13A protein affects ALS/FTD will be the next step. “How much is this gene actually contributing to the demise of motor neurons?” Zoghbi asked. Ma and colleagues found evidence that the UNC13A variants accelerate disease progression. Among 205 people from the Mayo Clinic bank who had FTD with TDP-43 deposits, those who were homozygous for a risk haplotype that includes both UNC13A risk variants died sooner than those with no risk variant.
Mis-spliced STMN2 and UNC13A might be just the tip of the iceberg. “I am certain there will be others,” Zoghbi said. “The data imply it [cryptic exons] could be relevant to any disorder with loss of nuclear TDP-43 function,” she added. Philip Wong, Johns Hopkins University, Baltimore, agreed. “Other cryptic exons could also be influenced by SNPs in introns that modify TDP-43 binding,” he wrote to Alzforum. In Wong's group, the hunt for them is on (full comment below).—Chelsea Weidman Burke
- In ALS and FTD, Two Different Routes to TDP-43 Aggregation
- CLIPs of TDP-43 Provide a Glimpse Into Pathology
- Does New Role for ALS-Linked Protein Help Explain Neurodegeneration?
- Beyond the Nucleus: TDP-43 Sticks Together, For Better or Worse
- Microtubule Regulator Connects TDP-43 to Axonal Dysfunction
- Massive ALS GWAS Cements Cytoskeletal Link to Disease
- Genetics Tie ALS into the Frontotemporal Dementia Spectrum
- Research Brief: Latest ALS GWAS Points to Loci on Chromosomes 9, 19
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