RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

Muscle proprioceptors in adult rat: mechanosensory signaling and synapse distribution in spinal cord.

The characteristic signaling and intraspinal projections of muscle proprioceptors best described in the cat are often generalized across mammalian species. However, species-dependent adaptations within this system seem necessary to accommodate asymmetric scaling of length, velocity, and force information required by the physics of movement. In the present study we report mechanosensory responses and intraspinal destinations of three classes of muscle proprioceptors. Proprioceptors from triceps surae muscles in adult female Wistar rats anesthetized with isoflurane were physiologically classified as muscle spindle group Ia or II or as tendon organ group Ib afferents, studied for their firing responses to passive-muscle stretch, and in some cases labeled and imaged for axon projections and varicosities in spinal segments. Afferent projections and the laminar distributions of provisional synapses in rats closely resembled those found in the cat. Afferent signaling of muscle kinematics was also similar to reports in the cat, but rat Ib afferents fired robustly during passive-muscle stretch and Ia afferents displayed an exaggerated dynamic response, even after locomotor scaling was accounted for. These differences in mechanosensory signaling by muscle proprioceptors may represent adaptations for movement control in different animal species.NEW & NOTEWORTHY Muscle sensory neurons signal information necessary for controlling limb movements. The information encoded and transmitted by muscle proprioceptors to networks in the spinal cord is known in detail only for the cat, but differences in size and behavior of other species challenge the presumed generalizability. This report presents the first findings detailing specializations in mechanosensory signaling and intraspinal targets for functionally identified subtypes of muscle proprioceptors in the rat.

A novel path to chronic proprioceptive disability with oxaliplatin: Distortion of sensory encoding.

Persistent neurotoxic side effects of oxaliplatin (OX) chemotherapy, including sensory ataxia, limit the efficacy of treatment and significantly diminish patient quality of life. The common explanation for neurotoxicity is neuropathy, however the degree of neuropathy varies greatly among patients and appears insufficient in some cases to fully account for disability. We recently identified an additional mechanism that might contribute to sensory ataxia following OX treatment. In the present study, we tested whether that mechanism, selective modification of sensory signaling by muscle proprioceptors might result in behavioral deficits in rats. OX was administered once per week for seven weeks (cumulative dose i.p. 70mg/kg) to adult female Wistar rats. Throughout and for three weeks following treatment, behavioral analysis was performed daily on OX and sham control rats. Compared to controls, OX rats demonstrated errors in placing their hind feet securely and/or correctly during a horizontal ladder rung task. These behavioral deficits occurred together with modification of proprioceptor signaling that eliminated sensory encoding of static muscle position while having little effect on encoding of dynamic changes in muscle length. Selective inability to sustain repetitive firing in response to static muscle stretch led us to hypothesize that OX treatment impairs specific ionic currents, possibly the persistent inward Na currents (NaPIC) that are known to support repetitive firing during static stimulation in several neuron types, including the class of large diameter dorsal root ganglion cells that includes muscle proprioceptors. We tested this hypothesis by determining whether the chronic effects of OX on the firing behavior of muscle proprioceptors in vivo were mimicked by acute injection of NaPIC antagonists. Both riluzole and phenytoin, each having multiple drug actions but having only antagonist action on NaPIC in common, reproduced selective modification of proprioceptor signaling observed in OX rats. Taken together, these findings lead us to propose that OX chemotherapy contributes to movement disability by modifying sensory encoding, possibly via a chronic neurotoxic effect on NaPIC in the sensory terminals of muscle proprioceptors.

Complex impairment of IA muscle proprioceptors following traumatic or neurotoxic injury.

The health of primary sensory afferents supplying muscle has to be a first consideration in assessing deficits in proprioception and related motor functions. Here we discuss the role of a particular proprioceptor, the IA muscle spindle proprioceptor in causing movement disorders in response to either regeneration of a sectioned peripheral nerve or damage from neurotoxic chemotherapy. For each condition, there is a single preferred and widely repeated explanation for disability of movements associated with proprioceptive function. We present a mix of published and preliminary findings from our laboratory, largely from in vivo electrophysiological study of treated rats to demonstrate newly discovered IA afferent defects that seem likely to make important contributions to movement disorders. First, we argue that reconnection of regenerated IA afferents with inappropriate targets, although often repeated as the reason for lost stretch-reflex contraction, is not a complete explanation. We present evidence that despite successful recovery of stretch-evoked sensory signaling, peripherally regenerated IA afferents retract synapses made with motoneurons in the spinal cord. Second, we point to evidence that movement disability suffered by human subjects months after discontinuation of oxaliplatin (OX) chemotherapy for some is not accompanied by peripheral neuropathy, which is the acknowledged primary cause of disability. Our studies of OX-treated rats suggest a novel additional explanation in showing the loss of sustained repetitive firing of IA afferents during static muscle stretch. Newly extended investigation reproduces this effect in normal rats with drugs that block Na(+) channels apparently involved in encoding static IA afferent firing. Overall, these findings highlight multiplicity in IA afferent deficits that must be taken into account in understanding proprioceptive disability, and that present new avenues and possible advantages for developing effective treatment. Extending the study of IA afferent deficits yielded the additional benefit of elucidating normal processes in IA afferent mechanosensory function.