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N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.

N(6)-Methyladenosine (m(6)A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m(6)A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m(6)A sites on mRNAs. FMRP depletion increased mRNA m(6)A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m(6)A-modified RNA targets.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.

SUMMARY 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., Graphical Abstract In Brief Edens et al. reveal fragile X mental retardation protein (FMRP) as an m6A reader that promotes the nuclear export of methylated mRNAs during neural differentiation. Loss of either Fmr1 or the m6A methyltransferase Mettl14 results in the nuclear accumulation of neural differentiation-related mRNAs, causing delayed neural progenitor differentiation in mice.

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in theSMA;p35−/−compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA modelin vivoand mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out oftauin SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degenerationin vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

Amyotrophic lateral sclerosis, or ALS for short, is a disease in which parts of the brain and spinal cord progressively degenerate. Specifically, the condition causes the nerve cells that control movement – called motor neurons – to die. As a result, people with ALS lose control of their muscles. The cause of ALS is not known, but evidence suggests that a person’s genetics plays a role in the development of the disease. Learning which genes are involved and what they do within cells may help scientists figure out what goes wrong in patients with ALS and how to treat the condition. People with ALS often experience an abnormal build up of proteins in their brain and spinal cord. Cells normally rely on molecules working together in the so-called ubiquitin proteasome system to eliminate unwanted proteins. A few mutations linked with ALS, and some other neurodegenerative conditions, have been traced back to genes encoding parts of this protein disposal system, which may help to explain the build up of proteins. However, our understanding of the genetic causes of the disease is far from complete. Now, Edens et al. report a new mutation in a gene that encodes a protein involved in the ubiquitin proteasome system. The gene, called UBQLN4, had not previously been linked to ALS, but looking at this gene in nearly 700 patients with ALS revealed a mutation in one patient with an inherited form of the disease. This mutation was not found in public databases that contain genetic information from tens of thousands of people without ALS. To better understand the effect of this newly identified mutation, Edens et al. recreated it in zebrafish embryos and motor neurons from mice. The mutation in UBQLN4 changed the shape of the cells in the spinal cords of the zebrafish and the mouse motor neurons. There was also a build up of excess proteins because the breakdown of proteins by the ubiquitin proteasome system was slowed. Specifically, there was an excess amount of a protein called beta-catenin, which is important for development and activity of the nervous system. Treating the mutant motor neuron cells with a drug called quercetin, which suppresses beta-catenin, reversed the defects seen in the cells. Larger studies are now needed to see how often this mutation occurs in patients with ALS and to determine if other forms of the disease might have a similar cause.

Summary 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.

Selective motor neuron degeneration is a hallmark of amyotrophic lateral sclerosis (ALS). Around 10 percent of all cases present as familial ALS (FALS), while sporadic ALS (SALS) accounts for the remaining 90 percent. Diverse genetic mutations leading to FALS have been identified, but the underlying causes of SALS remain largely unknown. Despite the heterogeneous and poorly understood etiology, different types of ALS exhibit overlapping pathology and common phenotypes, including protein aggregation and mitochondrial deficiencies. Here we review the current understanding of mechanisms leading to motor neuron degeneration in ALS, as they pertain to disrupted cellular clearance pathways, ATP biogenesis calcium buffering and mitochondrial dynamics. Through focusing on impaired autophagic and mitochondrial functions, we highlight how the convergence of diverse cellular processes and pathways contributes to common pathology in motor neuron degeneration.

Memory that food is inedible in Aplysia arises from training requiring three contingent events. Nitric oxide (NO) and histamine are released by a neuron responding to one of these events, attempts to swallow food. Since NO release during training is necessary for subsequent memory and NO substitutes for attempts to swallow, it was suggested that NO functions during training as a signal of attempts to swallow. However, it has been shown that NO may also be released in other contexts affecting feeding, raising the possibility that its role in learning is unrelated to signaling attempts to swallow. We confirmed that NO during learning signals attempts to swallow, by showing that a variety of behavioral effects on feeding of blocking or adding NO do not affect learning and memory that a food is inedible. In addition, histamine had effects similar to NO on learning that food is inedible, as expected if the transmitters are released together when animals attempt to swallow. Blocking histamine during training blocked long-term memory, and exogenous histamine substituted for attempts to swallow. NO also substituted for histamine during training. Histamine at concentrations relevant to learning activates neuron metacerebral cell (MCC). However, MCC activity is not a good monitor of attempts to swallow during training, since the neuron responds equally well to other stimuli. These findings support and extend the hypothesis that NO and histamine signal efforts to swallow during learning, acting on targets other than the MCC that specifically respond to attempts to swallow.

Summary Synaptic connections from a neuron onto itself (autapses) are not uncommon [1], but their contributions to information processing and behavior are not fully understood. Positive feedback mediated by autapses could in principle give rise to persistent activity, a property of some neurons in which a brief stimulus causes a long-lasting response [2]. We have identified an autapse that underlies a plateau potential causing persistent activity in the B31/B32 neurons of Aplysia . The persistent activity is essential to the ability of these neurons to initiate and maintain components of feeding behavior. Persistent activity in B31/B32 arises from a voltage-dependent muscarinic autapse and from pharmacologically identical network-based positive feedback. Depolarization via the autapse begins later than network-driven excitation, and the effect of the autapse is therefore overshadowed by the earlier network-based depolarization. In B31/B32 neurons isolated in culture only the autapse is present, and the autapse functionally replaces the missing network-based feedback. Properties of B31/B32 provide insight into a possible general function of autapses. Autapses might function along with synapses from presynaptic neurons as components of feedback loops.

SUMMARY Aplysia egg laying is a complex behavior requiring synchronized activity in many organs. Aspects of the behavior are synchronized viathe direct effects of peptide bag cell neurohormones and via stimuli arising during the behavior. Stimuli synchronizing egg laying were examined by treating A. fasciata with a nitric oxide (NO) donor. NO elicited normal appetitive and consummatory behaviors leading to the deposition of cordons containing egg capsules without eggs. The sites at which NO acts were investigated. The latency to egg deposition in response to a NO donor was shorter than that in response to other stimuli, consistent with NO acting at downstream sites from those affected by the other stimuli. The NO donor does not act on neurons in the head ganglia presynaptic to the bag cells or on the bag cells. Ligating the small hermaphroditic duct connecting the gonad to the accessory genital mass blocked egg laying in response to bag cell homogenates,but not in response to exogenous NO, indicating that NO does not act on the gonad. NO is released by transport of eggs along the small hermaphroditic duct, and NO directly acts on the accessory genital mass which packages eggs. NO also acts at a second site, independent of the effect on the accessory genital mass. A NO donor activates appetitive behaviors that normally precede egg laying even in A. californica that are unable to lay eggs.

Parkinson’s disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson’s disease or parkinsonian disorders. The pathogenesis of Parkinson’s disease remain largely elusive. Here, we report a novel genetic locus for an autosomal dominant, clinically typical and Lewy body confirmed Parkinson’s disease on the short arm of chromosome 20 (20pter-p12) and TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. The disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide the first genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson’s disease, with novel implications in understanding the pathogenic mechanism of Parkinson’s disease and for developing rational therapies.

Nitric oxide (NO) is widely used in neural circuits giving rise to learning and memory. NO is an unusual neurotransmitter in its modes of release and action. Is its association with learning and memory related to its unusual properties? Reviewing the literature might allow the formulation of a general principle on how NO and memory are related. However, other than confirming that there is indeed a strong association between NO and memory, no simple rules emerge on the role of NO in learning and memory. The effects of NO are not associated with a particular stage or form of memory and are highly dependent on species, strain, and behavior or training paradigm. Nonetheless, a review does provide hints on why NO is associated with learning and memory. Unlike transmitters acting via receptors expressed only in neurons designed to respond to the transmitter, NO is a promiscuous signal that can affect a wide variety of neurons, via many molecular mechanisms. In circuits giving rise to learning and memory, it may be useful to signal some events via a promiscuous messenger having widespread effects. However, each circuit will use the promiscuous signal in a different way, to achieve different ends.

mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Properties of a neuron may arise via endogenous mechanisms, or via interactions with other neurons. Culturing a neuron in isolation is a useful tool to distinguish between endogenous and circuit-derived properties. We identified two remarkable functional features of pattern initiator neurons B31/B32 in Aplysia when these neurons were cultured in isolation. These features were also present in situ, but were less prominent, and would have been missed had they not been observed first in the isolated cultured neurons. The properties are likely to be present in neurons of higher animals, but have not yet been observed. One feature was autaptic muscarinic self-excitation that contributes to the neuron's plateau potential, by which it initiates behavior. The other feature was the release of nitric oxide (NO) in the absence of spiking, which causes self-inhibition at rest. The nitrergic modulation of B31/B32 is likely to contribute to the control of feeding by dietary changes in the concentration of L: -arginine, the precursor from which NO is synthesized.

Background: Neural activity can be affected by nitric oxide (NO) produced by spiking neurons. Can neural activity also be affected by NO produced in neurons in the absence of spiking? Methodology/Principal Findings: Applying an NO scavenger to quiescent Aplysia buccal ganglia initiated fictive feeding, indicating that NO production at rest inhibits feeding. The inhibition is in part via effects on neurons B31/B32, neurons initiating food consumption. Applying NO scavengers or nitric oxide synthase (NOS) blockers to B31/B32 neurons cultured in isolation caused inactive neurons to depolarize and fire, indicating that B31/B32 produce NO tonically without action potentials, and tonic NO production contributes to the B31/B32 resting potentials. Guanylyl cyclase blockers also caused depolarization and firing, indicating that the cGMP second messenger cascade, presumably activated by the tonic presence of NO, contributes to the B31/B32 resting potential. Blocking NO while voltage-clamping revealed an inward leak current, indicating that NO prevents this current from depolarizing the neuron. Blocking nitrergic transmission had no effect on a number of other cultured, isolated neurons. However, treatment with NO blockers did excite cerebral ganglion neuron C-PR, a command-like neuron initiating food-finding behavior, both in situ, and when the neuron was cultured in isolation, indicating that this neuron also inhibits itself by producing NO at rest. Conclusion/Significance: Self-inhibitory, tonic NO production is a novel mechanism for the modulation of neural activity. Localization of this mechanism to critical neurons in different ganglia controlling different aspects of a behavior provides a mechanism by which a humeral signal affecting background NO production, such as the NO precursor L-arginine, could control multiple aspects of the behavior.

An increase in l-arginine hemolymph concentration acts as a postingestion signal inhibiting Aplysia feeding. At physiological concentrations (a 10-μM increase over background), the inhibitory effect of l-arginine is too weak to block feeding in hungry animals. However, a 10-μM increase in l-arginine concentration acts along with another inhibitory stimulus, the sustained presence of food odor, to inhibit feeding after a period of access to food. A physiological concentration of l-arginine also blocked the excitatory effect of a stimulus enhancing feeding, pheromones secreted by mating conspecifics. High concentrations of l-arginine (2.5 mM) alone also inhibited ad libitum feeding. l-arginine is the substrate from which nitric oxide synthase (NOS) produces nitric oxide (NO). Both an NO donor and a 10-μM increase in l-arginine inhibited biting in response to a weak food stimulus. Treatment with NOS inhibitors initiated food-finding and biting in the absence of food, indicating that food initiates feeding against a background of tonic nitrergic inhibition. Increased feeding in response to blocking NOS is accompanied by firing of the metacerebral (MCC) neuron, a monitor of food arousal. The excitatory effect on the MCC of blocking NOS is indirect. The data suggest that l-arginine acts by amplifying NO synthesis, which acts as a background stimulus inhibiting feeding. Background modulation of neural activity and behavior by NO may also be present in other systems, but such modulation may be difficult to identify because its effects are evident only in the context of additional stimuli modulating behavior.

Inhibiting nitric oxide (NO) synthesis during learning that food is inedible in Aplysia blocks subsequent memory formation. To gain insight into the function of NO transmission during learning we tested whether blocking NO synthesis affects aspects of feeding that are expressed both in a nonlearning context and during learning. Inhibiting NO synthesis with L-NAME and blocking guanylyl cyclase with methylene blue decreased the efficacy of ad libitum feeding. D-NAME had no effect. L-NAME also decreased rejection responses frequency, but did not affect rejection amplitude. The effect of L-NAME was explained by a decreased signaling that efforts to swallow are not successful, leading to a decreased rejection rate, and a decreased ability to reposition and subsequently consume food in ad libitum feeding. Signaling that animals have made an effort to swallow is a critical component of learning that food is inedible. Stimulation of the lips with food alone did not produce memory, but stimulation combined with the NO donor SNAP did produce memory. Exogenous NO at a concentration causing memory also excited a key neuron responding to NO, the MCC. Block of the cGMP second-messenger cascade during training by methylene blue also blocked memory formation after learning. Our data indicate that memory arises from the contingency of three events during learning that food is inedible. One of the events is efforts to swallow, which are signaled by NO by cGMP.