Microtubule Regulator Connects TDP-43 to Axonal Dysfunction

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.

Cryptic Splice Event. When TDP-43 goes missing, the splicing machinery messes up processing of stathmin 2 mRNA, incorporating an intron for an exon, and tacking on a premature poly-A tail, truncating the transcript (bottom). [Courtesy of Melamed et al., Nature Neuroscience.]

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.

Axonal Function. In a motor neuron (green), the microtubule-binding protein stathmin 2 (red) resides only in the Golgi apparatus (yellow) and axon growth cone, where it interfaces with F-actin bundles (white). [Courtesy of Klim et al., Nature Neuroscience.]

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

Comments

  1. I really like these papers. We always say that TDP-43 mislocalization is the "pathological hallmark of ALS," but the real pathological event that profoundly affects patients happens at the neuromuscular junction. Indeed, the dying back of the motor axons from the muscle is what actually leads to paralysis, and so the connections that these papers make between the loss of functional TDP-43, stathmin-2, and axonal integrity and regeneration mark an important advance to really getting at what this disease is all about from a clinical perspective. Of course, big questions remain about what the upstream initiator of TDP-43 dysfunction is in the vast majority of sporadic ALS cases, but at least now these papers convincingly point to one critical downstream consequence.

    View all comments by Krista Spiller

  2. It is encouraging that two different groups have come to very similar conclusions regarding stathmin 2. Therapeutically, one wonders whether antisense to modulate splicing of stathmin 2 might be useful. However, the stathmin 2 hypothesis is dependent on loss of TDP-43 function being the predominant pathogenic process in ALS, yet there is growing evidence that TDP-43 levels are increased in ALS, and that ALS linked TDP-43 mutations actually cause a gain of function rather than loss. More work needs to be done.

    View all comments by Jemeen Sreedharan

  3. While previous studies support the notion that repression of TDP-43 cryptic exons is compromised in neurodegenerative disease exhibiting TDP-43 pathology (Ling et al., 2015; Sun et al., 2017), including ALS, identification of direct targets of such a splicing defect that would impact on physiology of motor neurons has been elusive.

    Using human fibroblast- or iPSC-derived motor neurons, this pair of papers now provides compelling evidence to support stathmin-2 (STMN2), a protein regulating neuronal growth and enriched in motor neurons, as one such target. Upon depletion of TDP-43, Melamed and colleagues found incorporation into its pre-RNA a non-conserved cryptic exon containing a UG-rich TDP-43 binding element within intron 1 of STMN2. This non-conserved cryptic exon contained a poly(A) site, selection of which would lead to a truncated nonfunctional transcript; consequently, it is predicted that the level of mature STMN2 mRNA would be markedly diminished. They not only confirmed this prediction in neurons derived from trans-differentiation of ALS fibroblasts carrying mutant TDP-43, but also from motor cortex and spinal motor neurons from familial (C9ORF72) and sporadic ALS. A second prediction from such incorporation of STMN2 cryptic exon when TDP-43 was depleted would be a corresponding reduction in level of STMN2 protein. Using spinal cord tissues from ALS cases, Klim and colleagues showed that this prediction was nicely borne out. Finally, using an in vitro axotomy paradigm, these investigators validated the functional impact of losing STMN2 as a result of depleting TDP-43 in motor neurons. These data thus showed convincingly the critical role of TDP-43 in repressing non-conserved cryptic exons, particularly that of STMN2, in motor neurons of ALS.

    Despite these advances, an outstanding question is whether reduction in STMN2 upon deficits in TDP-43 splicing repression represents the major contributor to neurodegeneration in motor neurons of ALS patients. As opposed to STMN’s role in regenerative capacity of immature motor neurons, could other TDP-43 cryptic exon targets, such as those documented in both papers, contribute to neurodegeneration of mature, myelinated motor neurons in adults? While STMN2 can be an attractive target for development of ALS therapy, unraveling this issue in the future will provide critical information regarding its therapeutic potential as well as alternative strategies, including those that directly target TDP-43 splicing repression.

    References:

    Ling JP, Pletnikova O, Troncoso JC, Wong PC. NEURODEGENERATION. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. PubMed.

    Sun M, Bell W, LaClair KD, Ling JP, Han H, Kageyama Y, Pletnikova O, Troncoso JC, Wong PC, Chen LL. Cryptic exon incorporation occurs in Alzheimer's brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. Acta Neuropathol. 2017 Jun;133(6):923-931. Epub 2017 Mar 22 PubMed.

    View all comments by Philip Wong
    • User Profile Image Robert Baloh
      Director of Neuromuscular Medicine, Cedars-Sinai Medical Center
    • Posted:

    These are a pair of exciting papers that implicate loss of stathmin-2 as downstream of loss of TDP-43 that could be disruptive to neuronal health and contribute to neurodegeneration in ALS and FTD. The big question that remains, which is outside the scope of these studies, is whether loss of TDP-43 is a primary driver in ALS/FTD as opposed to toxic gain of function mechanisms, and if so how much stathmin-2 is responsible for neuronal damage versus the many other genes disrupted by loss of TDP-43 previously reported by this group and others. Regardless, these papers represent a very exciting and important step forward.

    View all comments by Robert Baloh

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References

News Citations

  1. CLIPs of TDP-43 Provide a Glimpse Into Pathology
  2. Friends of FUS: Protein's Many RNA Buddies Point to Disease
  3. Does New Role for ALS-Linked Protein Help Explain Neurodegeneration?

Paper Citations

  1. Highley JR, Kirby J, Jansweijer JA, Webb PS, Hewamadduma CA, Heath PR, Higginbottom A, Raman R, Ferraiuolo L, Cooper-Knock J, McDermott CJ, Wharton SB, Shaw PJ, Ince PG. Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones. Neuropathol Appl Neurobiol. 2014 Oct;40(6):670-85. PubMed.
  2. Riederer BM, Pellier V, Antonsson B, Di Paolo G, Stimpson SA, Lütjens R, Catsicas S, Grenningloh G. Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):741-5. PubMed.
  3. Stein R, Mori N, Matthews K, Lo LC, Anderson DJ. The NGF-inducible SCG10 mRNA encodes a novel membrane-bound protein present in growth cones and abundant in developing neurons. Neuron. 1988 Aug;1(6):463-76. PubMed.
  4. Morii H, Shiraishi-Yamaguchi Y, Mori N. SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons. J Neurobiol. 2006 Sep 1;66(10):1101-14. PubMed.
  5. D'Erchia AM, Gallo A, Manzari C, Raho S, Horner DS, Chiara M, Valletti A, Aiello I, Mastropasqua F, Ciaccia L, Locatelli F, Pisani F, Nicchia GP, Svelto M, Pesole G, Picardi E. Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS. Sci Rep. 2017 Aug 30;7(1):10046. PubMed.
  6. Krach F, Batra R, Wheeler EC, Vu AQ, Wang R, Hutt K, Rabin SJ, Baughn MW, Libby RT, Diaz-Garcia S, Stauffer J, Pirie E, Saberi S, Rodriguez M, Madrigal AA, Kohl Z, Winner B, Yeo GW, Ravits J. Transcriptome-pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS. Acta Neuropathol. 2018 Sep;136(3):405-423. Epub 2018 Jun 7 PubMed.
  7. Graf ER, Heerssen HM, Wright CM, Davis GW, DiAntonio A. Stathmin is required for stability of the Drosophila neuromuscular junction. J Neurosci. 2011 Oct 19;31(42):15026-34. PubMed.
  8. Duncan JE, Lytle NK, Zuniga A, Goldstein LS. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila. PLoS One. 2013;8(6):e68324. Print 2013 PubMed.

Further Reading

Primary Papers

  1. Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. 2019 Feb;22(2):167-179. Epub 2019 Jan 14 PubMed.
  2. Melamed Z, López-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, Freyermuth F, McMahon MA, Beccari MS, Artates JW, Ohkubo T, Rodriguez M, Lin N, Wu D, Bennett CF, Rigo F, Da Cruz S, Ravits J, Lagier-Tourenne C, Cleveland DW. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019 Feb;22(2):180-190. Epub 2019 Jan 14 PubMed.

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