RNA targets of TDP-43

In addition to searching for protein partners, we have sought to identify mRNA targets of TDP-43 in neurons.  We recently showed that TDP-43 regulates the localization and translation of futsch/MAP1B mRNA in Drosophila. The defects we identified in the ALS model fly motor neurons are remarkably similar to those found in human ALS spinal cord neurons (Figure 2). This is significant because on one hand, dysregulation of Futsch/MAP1B offers an explanation for the microtubule and synaptic instability found in ALS and on the other, it provides proof of principle that our “fly-to-man” approach can be effective in elucidating the pathomechanism of neurodegeneration.


Figure 2. Microtubule-stabilizing protein Futsch/MAP1B is retained in motor neuron cell bodies. Top panels: motor neurons showing increased protein in ALS flies (top right) compared to controls (top left). White arrowheads indicate motor neuron cell bodies. Bottom panels: spinal cords from ALS patients (bottom right) show higher levels of Futsch/MAP1B compare to controls (bottom left). Black arrowheads indicate motor neuron cell bodies.

 

 

To identify neuronal specific mRNA targets and to determine what stage of translation is impacted by TDP-43 expression, we are using RNA immunoprecipitations (RIP) and translating ribosome affinity purification (TRAP). Using RIP and TRAP, we have identified several novel candidate translational targets including hsc70-4 mRNA, which encodes a molecular chaperone that controls synaptic vesicle (SV) trafficking, as well as additional candidates implicated in synaptic function. Notably, restoring Hsc70-4 levels in motor neurons by overexpression rescues synaptic vesicle endocytosis defects caused by ALS-associated mutant TDP-43 (TDPG298S).

Figure 3. A model for TDP-43 and Hsc70-4 interactions. (A) In controls, TDP-43 does not sequester mRNA targets or protein partners leading to normal levels of mRNA translation and synaptic proteins at the NMJ. As a result, SVC occurs as normal. (B) Motor neuron expression of TDP-43 results in decreased synaptic expression of Hsc70-4 and defects in SV endocytosis. Mutant TDP-43 sequesters hsc70-4 mRNA in insoluble complexes and inhibits its translation. 

 

These findings suggest that ALS is caused at least in part by dysregulation of translation of key mRNAs regulating the synaptic vesicle cycle at the neuromuscular junction, consistent with ALS being “a synaptopathy”. The identification of hsc70-4 mRNA, a key cellular chaperone as a translational target of disease-associated mutant TDP-43 protein and a component of TDP-43 aggregates, is exciting because it unifies the ribostasis (i.e., RNA processing) and proteostasis (i.e., protein folding) hypotheses of neurodegeneration.

We are currently investigating additional mRNA candidate targets encoding among others synaptic and metabolic proteins.