Escort Service: A Cytoplasmic Role for TDP-43

TDP-43 regulates mRNA metabolism in the nucleus and occurs in the cytoplasm mainly in the form of aggregates in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and other neurodegenerative disorders. Researchers have debated whether this RNA binding protein performs any physiological role outside the nucleus, and now a new study suggests that it does. In the February 5 Neuron, J. Paul Taylor, St. Jude Children’s Research Hospital, Memphis, Tennessee, and colleagues report that TDP-43 helps move mRNA-containing granules along axons to nerve terminals. Pathogenic mutations in TDP-43 hold back this traffic. “This paper defines a novel function for TDP-43, and provides a mechanism by which mutations could cause dysfunction,” said Aaron Gitler, Stanford University School of Medicine, California, who was not involved in the study.

TDP-43, or TAR DNA-binding protein 43, is best known for its hand in DNA transcription and RNA splicing in the nucleus. However, scientists have also seen it binding to proteins responsible for the transport of mRNAs in the cytoplasm, and it has been spotted along dendrites and axons all the way out to their tips (see Freibaum et al., 2010; Narayanan et al., 2012). This hints that TDP-43 helps ferry certain mRNAs to the far reaches of neurons, where they can be translated rapidly in situ in response to neuronal activity (see Liu-Yesucevitz et al., 2011). However, no one has observed TDP-43 behave in this fashion. First authors Nael Alami, Rebecca Smith, and colleagues set out to explore that possibility by comparing the behavior of wild-type and mutant versions of the human protein in cells from flies, mice, and people.

In motor neurons from flies and primary cortical neurons from mice, the researchers fluorescently tagged overexpressed wild-type human TDP-43 or one of two ALS-causing mutant forms—M337V or A315T. Most of the wild-type TDP-43 stayed in the nucleus, though a steady trickle of cytoplasmic granules containing the protein extended down to the tips of the axon (see image below). In contrast, mutant forms of the protein built up in and around the cell body; little to none of it made it very far along the axon. Live imaging revealed that granules containing mutant TDP-43 moved more haltingly down the axon than the native form. Forward motion was particularly sluggish, and a higher percentage of the granules moved backward. Interestingly, mitochondria moved equally well down the axon in all mice, suggesting the TDP-43 mutants did not impair axonal transport in general.

Going Places: A complex containing TDP-43 (red) moves along the axon (white outline) of a mouse cortical neuron. [Image courtesy of Neuron, Alami et al., 2014 Figure 2A]

To test whether TDP-43 mutations limit transport of mRNAs, Alami and colleagues created an “mRNA beacon.” This is a fluorescent reporter that allowed them to follow transcripts of Neurofilament-L (Nefl), a well-known TDP-43 binding partner. Most previous attempts to label mRNA required genetic modifications or chemical treatment that killed the cells. In this case, the researchers were able to measure transcripts in live cells by adding a fluorescent oligonucleotide that bound specifically to the Nefl transcript. The beacon tracked together with Green Fluorescent Protein-labeled TDP-43. In mouse cortical neurons, Nefl-positive granules containing TDP-43 moved forward, on average, while those without TDP-43 moved back toward the nucleus, suggesting that the RNA-binding protein helps drive anterograde motion of the granules. The researchers next tracked the movement of human Nefl transcript in motor neurons cultured from the induced pluripotent stem cells of three ALS patients. These cells expressed endogenous levels of TDP-43 with the M337V, A315T, or G298S mutations. The scientists saw that anterograde motion of Nefl was impaired compared with that in cells from healthy people.

The work sheds light on yet another way in which TDP-43 dysfunction could lead to ALS and FTLD, according to the authors. “We think that TDP-43 is responsible for the movements of its mRNA partners along the axon. This might explain why motor neurons, which have especially long axons, are selectively affected in ALS,” said Alami. He and colleagues plan to test if these trafficking alterations translate to problems with protein synthesis at the synapse, neural development, or neurodegeneration.

In addition, Alami’s group plans to make other mRNA beacons to track transcripts of FUS and granulin, which are associated with ALS, to see if TDP-43 mutations affect their transport. The scientists also want to know whether disease-causing mutations in other RNA-binding proteins disrupt delivery of mRNA along the axon.

Wilfried Rossoll, Emory University, Atlanta, has previously reported that TDP-43 is transported down the axons of motoneurons (Fallini et al., 2012). He called this new study exciting for taking a rare look at the axonal function of TDP-43 and finding similar effects in several disease models. It will be important to see whether this transport defect applies to TDP-43 proteinopathies that involve mutations in other proteins and in sporadic forms of ALS, he added. Gitler, who penned an accompanying Preview in Neuron, noted that this mRNA transport problem is likely one of several defects that arise when TDP-43 is mutated. Genetic disruptions also cause loss of nuclear function and toxic aggregation in the cytoplasm. It is unclear which contributes most to disease, he said.

References:

Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, Kiskinis E, Winborn B, Freibaum BD, Kanagaraj A, Clare AJ, Badders NM, Bilican B, Chaum E, Chandran S, Shaw CE, Eggan KC, Maniatis T, Taylor JP. Axonal Transport of TDP-43 mRNA Granules Is Impaired by ALS-Causing Mutations. Neuron. 2014 Feb 5;81:536-543. [Pubmed].

Jovičić A & Gitler AD. TDP-43 in ALS: Stay on Target…Almost There. Neuron. 2014 Feb 5;81. [Pubmed].


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