Nearly all cases of amyotrophic lateral sclerosis, and half of frontotemporal dementia ones, are marked by deposits of the RNA-binding protein TDP-43, yet researchers still have little idea how this pathology relates to neurodegeneration. Now, two independent studies in the January 14 Nature Neuroscience implicate a single TDP-43 target. Researchers led by Kevin Eggan at Harvard University, and a separate group led by Don Cleveland at the University of California, San Diego, and Clotilde Lagier-Tourenne at Massachusetts General Hospital, both report that a lack of nuclear TDP-43 causes faulty splicing and loss of stathmin 2 mRNA. This protein regulates microtubules and promotes neurite growth. Without it, cultured motor neurons were unable to extend or repair axons. In ALS, motor axons become disconnected from muscle fiber, triggering neuron degeneration.
- In human motor neurons, loss of nuclear TDP-43 suppressed expression of stathmin 2.
- Loss of this cytoskeletal regulator slowed axon growth and repair.
- STMN2 is low in sporadic and familial ALS patients.
Krista Spiller at the University of Pennsylvania in Philadelphia said the papers offer valuable insight. “The dying back of motor axons from muscle is what leads to paralysis, and so the connections these papers make between loss of functional TDP-43, stathmin-2, and axonal integrity and regeneration mark an important advance toward understanding this disease from a clinical perspective,” she wrote to Alzforum (full comment below).
Does the TDP-43 effect seen in cell culture actually happen in ALS, however? Both groups of researchers reported less stathmin 2 (STMN2) in spinal cord tissue from ALS patients than in control tissue. This was the case for sporadic disease as well as that caused by the C9ORF72 expansion, hinting it could represent a general mechanism. “These two fascinating papers raise the possibility that myriad causes of ALS could be ameliorated by restoring the expression of one key axonal protein, providing hope for a common therapy,” Ron Klein at Louisiana State University Health Sciences Center in Shreveport wrote to Alzforum.
Previously, numerous studies linked the loss of nuclear TDP-43 to widespread RNA mis-splicing (Mar 2011 news; Oct 2012 news; Highley et al., 2014). Philip Wong at Johns Hopkins University in Baltimore had found that the RNA-binding protein prevents the splicing machinery from mistaking pieces of introns for exons; without TDP-43’s guidance, numerous such “cryptic exons” are retained in mRNAs, leading to cellular dysfunction and death (Aug 2015 news). However, these studies did not identify specific mis-splicing events associated with ALS pathology.
Eggan and colleagues looked for affected transcripts by knocking down TDP-43 in human motor neurons derived from induced pluripotent stem cells. First authors Joseph Klim and Luis Williams found the knockdown altered levels of 885 transcripts, and affected splicing of another 815. There were only 50 transcripts in common between the two sets. One of these was stathmin 2. Neurons lacking TDP-43 made about half as much STMN2 as did control neurons, and the transcript contained a cryptic exon that truncated the mRNA just after exon 1.
Cleveland and colleagues provided more detail on how this occurs. First author Ze’ev Melamed knocked down TDP-43 in a neuronal cell line and saw the biggest expression change in STMN2, with its mRNA levels squelched by 85 percent. Sequencing the transcript, the authors also found the extra exon right after exon 1, which they dubbed 2a. They found that it had a polyadenylation site at its end, causing the splicing machinery to terminate the transcript there and tack on a poly-A tail (see image above). Thus, this transcript cannot make functional STMN2.
A lack of TDP-43 inhibits STMN2 production, but what about TDP-43 mutations known to cause ALS? Eggan and colleagues found less STMN2 in motor neurons made from people with the M337V TDP-43 variant, while Cleveland and colleagues detected STMN2 suppression in neurons carrying the N352S mutation. In addition, Eggan’s group induced TDP-43 to mislocalize from nucleus to cytosol, and again found low STMN2. The findings suggest that many different perturbations in TDP-43 can curtail STMN2 production.
The researchers next examined the consequences of STMN2 loss for motor neurons. Normally, the protein is massively expressed in these cells, nearly as abundant as neurofilaments. Several studies have found STMN2, previously called SCG10, to be essential for neurite extension (Riederer et al., 1997; Stein et al., 1988; Morii et al., 2006). In keeping with this, Eggan and colleagues detected STMN2 in axon growth cones of human motor neurons (see image below). Knocking down STMN2 suppressed neurite growth in healthy cells and slowed axon regrowth after injury, as did knocking down TDP-43. Likewise, Cleveland and colleagues reported a 90 percent suppression of axonal regeneration after STMN2 loss. Conversely, boosting STMN2 levels rescued neurite growth and repair in both studies.
To tie the findings to ALS cases, Eggan and colleagues examined postmortem spinal cord tissue from three people with ALS, finding about half the level of STMN2 protein as in three healthy controls. They also analyzed published expression data from ALS spinal cord, and identified the presence of the cryptic exon in five of six ALS samples but not in control samples (D’Erchia et al., 2017).
Cleveland and colleagues analyzed a different dataset, in which RNA was isolated from lumber motor neurons microdissected from human spinal cord (Krach et al., 2018). They found the STMN2 cryptic exon in all 13 sporadic ALS patients, but not in the seven healthy controls. In addition, patient samples contained about half as much STMN2 transcript as controls. Cleveland and colleagues also extracted RNA from postmortem thoracic spinal cord of three ALS patients carrying the C9ORF72 expansion, and three with an SOD1 mutation. The former accumulate TDP-43 while the latter do not. Notably, C9ORF72 samples contained the cryptic exon and SOD1 samples did not.
How relevant to pathology is STMN2 loss? One clue comes from Drosophila. Studies reported disruption of the neuromuscular junction and paralysis in flies lacking stathmin (Graf et al., 2011; Duncan et al., 2013). There is no comparable mouse data, but Cleveland and colleagues report that rodent STMN2 does not contain the cryptic polyadenylation site, and alternative splicing does not occur in these animals.
“Our evidence supports development of therapeutic strategies for ALS, FTD, and other neurodegenerative diseases affected by TDP-43 proteinopathy through restoration of stathmin 2,” Cleveland and colleagues proposed. Jemeen Sreedharan at King’s College London agreed this might be worth trying, but noted that some studies have found TDP-43 levels up in ALS, rather than down, and have reported mutations believed to cause a gain of function rather than a loss (full comment below). However, Eggan’s data suggest these findings could still be compatible with STMN2 suppression. When they overexpressed TDP-43 in human motor neurons, they again found low STMN2, suggesting that either too much or too little of the protein disrupts expression of the microtubule regulator.—Madolyn Bowman Rogers
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