Small molecule activation of m6A mRNA methylation as a novel approach for neuroprotection

Abstract

N6-Methyladenosine (m6A) is the most common mRNA base modification in eukaryotes. Methylation of adenosine residues to m6A contributes to the regulation of splicing, transport, stability, and translation of mRNA and two main classes of enzymes regulate it. The formation of m6A is catalysed by a methyltransferase complex containing methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumour 1-associated protein (WTAP) as well as monomeric METTL16. Demethylation of m6A is catalysed by the fat mass and obesity-associated protein FTO and the RNA demethylase AlkB homolog 5 (ALKBH5). The m6A mRNA methylation dysregulation occurs in the nervous system and in Parkinson's disease (PD), but it remains poorly studied. Moreover, the role of m6A mRNA methylation in neuronal survival, neuroprotection, and neuroregeneration is unclear. We have earlier used high-throughput virtual screening of large compound libraries and identified four unique small-molecule ligands that activate m6A mRNA methylation by binding to the METTL3/14/WTAP complex and enhancing the binding of the methylation substrate SAM to nanomolar concentrations. Following this, we now discovered that two methyltransferase activators at 10 nM concentrations supported the survival and protected dopamine (DA) neurons in culture in growth factor deprivation and 6-hydroxydopamine (6-OHDA) neurotoxin models. In contrast, METTL3/14 inhibitor STM2457 triggered death of DA neurons. For clinical translation we also tested the most efficient compound C4 on induced pluripotent stem cell-derived human DA neurons and in animal model of Parkinson's disease (PD). C4 compound protected human DA neurons from 6-OHDA-induced cell death and increased neurite outgrowth and the number of processes demonstrating that it has both neuroprotective and neurorestorative properties. METTL3/14 activator C4 improved motor behaviour and protected DA neurons and their fibres faster and much more efficiently than GDNF in the rat 6-OHDA model of PD. These are the first specific activators of METTL3/14/WTAP and first demonstration that m6A regulators can protect and regenerate neurons. These data demonstrate that m6A mRNA methylation is a novel pathway regulating neuronal survival and regeneration.

Effect of RNA m6A methyltransferase activation by a low molecular weight compound on anxiety- and depression-related behaviours, monoamine neurochemistry and striatal gene expression in the rat

“…To extend knowledge beyond the monoamine systems and to analyse what mRNAs are regulated by METTL3/METTL14 activator CHMA1004 we also performed RNA sequencing in striatum in female rats.The low and high dose of CHMA1004 resulted in a different landscape of differential gene expression, suggesting that response to subchronic drug treatment involved dose-dependent adaptive changes that had also been apparent in behavioural and biochemical effects; however, expression of several genes was altered in a similar manner. Many effects of CHMA1004 on gene expression agree with what is known of m 6 A in neurodegeneration and affective disorders (see Introduction), and the capacity of the drug to protect dopamine neurons (Yu et al, 2023) as well as its behavioural effects in the present study. For example, Galns and Samd4a have been implicated in neuronal viability and plasticity; Galns that encodes a lysosomal exohydrolase has been considered a gene related to Parkinson’s disease (Wettergren et al, 2012) and Samd4a is a RNA binding protein that can bind to a variety of target mRNAs through stem-loop structures, also known as Smaug recognition elements (SREs).…”

“…Inhibition of m 6 A RNA demethylases Fto and Alkbh5, which results in increased m6A levels, can support the survival of dopamine neurons (Selberg et al, 2021). Furthermore, the METTL3/METTL14 activating compound CHMA1004 was also found to support survival of cultured dopamine neurons that were deprived of growth factors and to protect neurons from 6-OHDA toxicity (Yu et al, 2023). Importantly, intrastriatal administration of CHMA1004 improved motor behaviour and increased tyrosine hydroxylase positive fibre density in mice after 6-OHDA lesion; it also protected human cultured dopamine neurons in that study.…”

“…Importantly, CHMA1004 (methyl-piperazine-2- carboxylate) protected stem cell-derived human dopamine neurons against 6-OHDA induced cell death. Furthermore, intrastriatal administration of CHMA1004 attenuated amphetamine-induced ipsilateral rotations after 6-OHDA treatment and increased dopamine neuron fibre density in the rat model of PD (Yu et al, 2023). Given the massive arborization of dopaminergic axons (Matsuda et al, 2009), dopaminergic neural projections may be particularly sensitive to the epitranscriptomic regulation of mRNA translation that occurs in the axonal and dendritic ribosomes (Biever et al, 2020).…”