m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

SMN Is Physiologically Downregulated at Wild-Type Motor Nerve Terminals but Aggregates Together with Neurofilaments in SMA Mouse Models.

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of ß-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.

FMRP Modulates Neural Differentiation through m6A-Dependent mRNA Nuclear Export.

N6-methyladenosine (m6A) modification of mRNA is emerging as a vital mechanism regulating RNA function. Here, we show that fragile X mental retardation protein (FMRP) reads m6A to promote nuclear export of methylated mRNA targets during neural differentiation. Fmr1 knockout (KO) mice show delayed neural progenitor cell cycle progression and extended maintenance of proliferating neural progenitors into postnatal stages, phenocopying methyltransferase Mettl14 conditional KO (cKO) mice that have no m6A modification. RNA-seq and m6A-seq reveal that both Mettl14cKO and Fmr1KO lead to the nuclear retention of m6A-modified FMRP target mRNAs regulating neural differentiation, indicating that both m6A and FMRP are required for the nuclear export of methylated target mRNAs. FMRP preferentially binds m6A-modified RNAs to facilitate their nuclear export through CRM1. The nuclear retention defect can be mitigated by wild-type but not nuclear export-deficient FMRP, establishing a critical role for FMRP in mediating m6A-dependent mRNA nuclear export during neural differentiation.

Impact of energetic neutral nitrogen atoms created by glow discharge air plasma on the physico-chemical and rheological properties of kithul starch.

The changes in physico-chemical and rheological properties of kithul starch by the impact of energetic neutral nitrogen atoms produced by the glow discharge air plasma with novel technique were analysed. Here, treatment was carried out at different power levels (5 W & 15 W) and treatment time (30 min & 60 min). Decrease in amylose and moisture content and increase in swelling index and solubility of plasma treated kithul starch were observed. Fourier transform infrared spectra of plasma treated starch showed variation in the peaks corresponding to CH2 and OH groups. Relative crystallinity significantly (p ≤ 0.05) decreased after plasma treatment. Scanning electron microscopy showed severe damages on the starch granular surface by the effect of energetic neutral nitrogen atoms. Pasting properties increased and thermal properties showed significant (p ≤ 0.05) changes after plasma treatment. Storage modulus (G') and loss modulus (G″) reduced at higher levels of plasma power and it indicated to its weak gel formation.

Regulation of Survival Motor Neuron Protein by the Nuclear Factor-Kappa B Pathway in Mouse Spinal Cord Motoneurons.

Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach.

Smn-Deficiency Increases the Intrinsic Excitability of Motoneurons.

During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease.

Synaptotagmin-2, and -1, linked to neurotransmission impairment and vulnerability in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the most frequent genetic cause of infant mortality. The disease is characterized by progressive muscle weakness and paralysis of axial and proximal limb muscles. It is caused by homozygous loss or mutation of the SMN1 gene, which codes for the Survival Motor Neuron (SMN) protein. In mouse models of the disease, neurotransmitter release is greatly impaired, but the molecular mechanisms of the synaptic dysfunction and the basis of the selective muscle vulnerability are unknown. In the present study, we investigated these open questions by comparing the molecular and functional properties of nerve terminals in severely and mildly affected muscles in the SMNΔ7 mouse model. We discovered that synaptotagmin-1 (Syt1) was developmentally downregulated in nerve terminals of highly affected muscles but not in low vulnerable muscles. Additionally, the expression levels of synaptotagmin-2 (Syt2), and its interacting protein, synaptic vesicle protein 2 (SV2) B, were reduced in proportion to the degree of muscle vulnerability while other synaptic proteins, such as syntaxin-1B (Stx1B) and synaptotagmin-7 (Syt7), were not affected. Consistently with the extremely low levels of both Syt-isoforms, and SV2B, in most affected neuromuscular synapses, the functional analysis of neurotransmission revealed highly reduced evoked release, altered short-term plasticity, low release probability, and inability to modulate normally the number of functional release sites. Together, we propose that the strong reduction of Syt2 and SV2B are key factors of the functional synaptic alteration and that the physiological downregulation of Syt1 plays a determinant role in muscle vulnerability in SMA.