NMDAR mediated dynamic changes in m6A inversely correlates with neuronal translation.

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (m6A) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of m6A modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of m6A modification in response to NMDAR stimulation. We have consistently observed that 5 min NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the m6A signal at 5 min time point, correlating with the global translation inhibition. The NMDAR induced increase in the m6A signal is accompanied by the redistribution of the m6A marked RNAs from translating to the non-translating pool of ribosomes. The increased m6A levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in m6A levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization.

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

The Role of Dynamic miRISC During Neuronal Development.

Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.

Differential Regulation of Syngap1 Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity.

SYNGAP1, a Synaptic Ras-GTPase activating protein, regulates synapse maturation during a critical developmental window. Heterozygous mutation in SYNGAP1 (SYNGAP1 -/+) has been shown to cause Intellectual Disability (ID) in children. Recent studies have provided evidence for altered neuronal protein synthesis in a mouse model of Syngap1 -/+. However, the molecular mechanism behind the same is unclear. Here, we report the reduced expression of a known translation regulator, FMRP, during a specific developmental period in Syngap1 -/+ mice. Our results demonstrate that FMRP interacts with and regulates the translation of Syngap1 mRNA. We further show reduced Fmr1 translation leads to decreased FMRP level during development in Syngap1 -/+ which results in an increase in Syngap1 translation. These developmental changes are reflected in the altered response of eEF2 phosphorylation downstream of NMDA Receptor (NMDAR)-mediated signaling. In this study, we propose a cross-talk between FMRP and SYNGAP1 mediated signaling which can also explain the compensatory effect of impaired signaling observed in Syngap1 -/+ mice.