Sujata Dalal

Introduction. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with characteristic progressive upper and lower motor neuron degeneration.^1,2 It has a poor prognosis with only two drugs, riluzole and edaravone, being prescribed for treatment.2 In most ALS cases, the affected motor neurons are afflicted with nuclear loss and cytoplasmic accumulation of the RNA binding protein transactive response DNA-binding protein 43 (TDP-43).3 TDP-43 is key in RNA metabolism, but how exactly it’s loss can cause neurodegeneration is unclear.3 One explanation is seen in how mRNA transcripts for stathmin-2, a protein that stimulates microtubule instability required for axonal outgrowth and repair, are reduced in TDP-43 depleted neurons.3,4 The mechanism behind how TDP-43 reduces stathmin-2 mRNA transcripts and how this decrease leads to the hallmark motor neurodegeneration seen in ALS can be understood and targeted for future therapeutic developments.³ Methods. TDP-43 function was manipulated in a human neuronal cell line, SH-SY5Y, by using short interfering RNA (siRNA) to deplete TDP-43 and by using CRISPR Cas9 to introduce an ALS-causing mutation into both endogenous TDP-43 gene loci.³ Overall, lowered TDP-43 levels showed a reduction of stathmin-2 mRNA  and protein which was confirmed by quantitative PCR (qPCR) and immunoblotting.³ RNA-Seq showed that depletion or mutation in TDP-43 induced a new spliced exon, called exon 2a, within intron 1.³ RT-PCR and qPCR showed altered splicing and ligation of exon 1 to exon 2a in the manipulated SH-SY5Y cells.³ By using anchored oligo(dT) primers for RT-PCR, it was confirmed that the incorporation of exon 2a causes alternative polyadenylation in the normal intron 1.³ TDP-43 or stathmin-2 suppressed iPSC-derived motor neurons were transduced with a lentivirus that restored stathmin-2 levels which influenced regenerative capability.³ Results. Experimentally depleted levels of TDP-43 resulted in decreased stathmin-2 expression.³ Decreased levels of nuclear TDP-43 reduced binding to stathmin-2 pre-mRNA, which revealed a cryptic polyadenylation site that generated non-functional mRNA and truncated stathmin-2.³ Reintroduction of stathmin-2 restored axonal growth and regeneration.³ Conclusion. TDP-43 pathology is a hallmark of ALS.³ Nuclear loss of TDP-43 results in exposure of a cryptic exon through loss of RNA binding.⁵ The cryptic exon generates truncated stathmin-2 mRNA producing a nonfunctional protein.⁵ The nonfunctional stathmin-2 provides the mechanistic link for how TDP-43 pathology can result in the neurodegeneration seen in ALS.³ Since stathmin-2 is an important axonal maintenance factor and its loss decreases microtubule outgrowth in motor neurons, increasing its expression can serve as a potential therapeutic for ALS.⁵

1. Theunissen F, West PK, Brennan S, et al. New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis. Transl Neurodegener. 2021;10(1):46.
2. Hardiman O, Al-Chalabi A, Chio A, et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers. 2017;3:17085.
3. Melamed Z, López-Erauskin J, Baughn MW, et al. Premature polyadenylation- mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019;22(2):180-190.
4. Benarroch E. What Is the Role of Stathmin-2 in Axonal Biology and Degeneration? [published correction appears in Neurology. 2022 Mar 1;98(9):384]. Neurology. 2021;97(7):330-333. doi:10.1212/WNL.0000000000012419
5. Klim JR, Williams LA, Limone F, et al. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. 2019;22(2):167-179.