METTL3-mediated regulation of motor neuron function

SUMMARY Dementing neurodegenerative diseases affect millions of people and have an annual cost exceeding $380 billion dollars in the USA alone. To gain insights into potential effective therapeutic strategies for these incurable disorders, herein we focused our attention on the RNA-binding protein TAR DNA-binding protein-43 (TDP-43), whose altered subcellular distribution and role are thought to contribute to a number of dementing disorders including Alzheimer’s disease (AD) and the clinical spectrum of amyotrophic lateral sclerosis-frontotemporal dementia (ALS/FTD). TDP-43, like other RNA-binding proteins with low-complexity domains are involved in the formation of membrane-less organelles associated with RNA processing via liquid-liquid phase separation (LLPS) assembly. While disease-associated mutations in TDP-43 can disrupt LLPS and its ensuing roles, how wild-type TDP-43 subcellular distribution and function are altered in the majorly of AD and ALS/FTD cases, which are gene mutation negative, remain enigmatic. We propose, herein, to investigate the role of post-transcriptional regulatory mechanisms of TDP-43 mediated by N6-methyladenosine (m6A). This idea stems from our finding that TARDBP, the gene encoding for TDP-43, is among the m6A targets in embryonic stem cell-derived motor neurons (ES-MNs) and that its expression is under m6A epitranscriptomic regulation. Our central hypothesis is that loss of m6A marks on TDP-43 causes changes in its expression and in its subcellular distribution and role, which, in turn, may contribute to neurodegeneration. The rationale for this research is that, once it is known how m6A-mediated regulation of TDP-43 contributes to neurodegeneration, innovative genetic or pharmacological strategies can be devised for the treatment of these incurable human diseases. We propose to first (AIM 1) characterize the spatiotemporal susceptibility of MNs to m6A loss. Accordingly, we will define the pattern of MN loss in both spinal cord and brainstem in mice with Mettl3 depletion at both presymptomatic and symptomatic stages using a battery of behavioral, physiological and morphological techniques. Then, since our preliminary data show that TARDBP levels are increased when m6A is lost, in AIM 2, we will determine how the loss of m6A impairs TDP-43 expression and its subcellular localization. Our preliminary data also show that m6A depletion in ES-MNs results in reduced survival and neurite outgrowth. Thus, in AIM 3, we will provide a causal link between m6A-dependent dysregulation of TDP-43 and neurodegeneration by using CRISPR-Cas-based site-specific m6A editing of TDP-43 mRNA. Our work is expected to elucidate the role of m6A modification on TDP-43 subcellular distribution and functions. Thus, our findings will have a positive impact by advancing our mechanistic understanding of TDP-43 requirement for neuronal survival and by providing new targets for epitranscriptome- modulating therapies for age-related TDP-43 proteinopathies.