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Motor neurons are a remarkably powerful cell type in the central nervous system. They innervate and control the contraction of virtually every muscle in the body and their dysfunction underlies numerous neuromuscular diseases. Some motor neurons seem resistant to degeneration whereas others are vulnerable. The intrinsic heterogeneity of motor neurons in adult organisms has remained elusive. The development of high-throughput single-cell transcriptomics has changed the paradigm, empowering rapid isolation and profiling of motor neuron nuclei, revealing remarkable transcriptional diversity within the skeletal and autonomic nervous systems. Here, we discuss emerging technologies for defining motor neuron heterogeneity in the adult motor system as well as implications for disease and spinal cord injury. We establish a roadmap for future applications of emerging techniques - such as epigenetic profiling, spatial RNA sequencing, and single-cell somatic mutational profiling to adult motor neurons, which will revolutionize our understanding of the healthy and degenerating adult motor system.

Neurons born in the embryo can undergo a protracted period of maturation lasting well into postnatal life. How gene expression changes are regulated during maturation and whether they can be recapitulated in cultured neurons remains poorly understood. Here, we show that mouse motor neurons exhibit pervasive changes in gene expression and accessibility of associated regulatory regions from embryonic till juvenile age. While motifs of selector transcription factors, ISL1 and LHX3, are enriched in nascent regulatory regions, motifs of NFI factors, activity-dependent factors, and hormone receptors become more prominent in maturation-dependent enhancers. Notably, stem cell-derived motor neurons recapitulate ~40% of the maturation expression program in vitro, with neural activity playing only a modest role as a late-stage modulator. Thus, the genetic maturation program consists of a core hardwired subprogram that is correctly executed in vitro and an extrinsically-controlled subprogram that is dependent on the in vivo context of the maturing organism.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease. Blum et al. performed single-nucleus RNA sequencing of the adult mouse spinal cord. This analysis revealed heterogeneity in the autonomic and skeletal motor systems and provides a resource to study motor neurons in health and disease.

Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2–100 μm, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μm. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nm) to very low (>100 μm) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

A structural model is proposed for the processing of interaural intensity differences by the lateral superior olive. One fundamental assumption is that the incoming excitatory projections from the ipsilateral anteroventral cochlear nucleus innervate columns of LSO neurons serially according to threshold. A second fundamental assumption is that the inhibitory innervation from the ipsilateral medial nucleus of the trapezoid body is also serially arranged according to threshold but in the opposite direction along the LSO column. Using neurophysiological and neuroanatomical data for neuronal response curves, connectional patterns, and cell and synapse numbers, the model was formulated quantitatively and implemented for machine computation. Azimuthal location is encoded by the position along the LSO column where LSO cell firing first goes to zero. Accuracy of coding was tested for three different connectional schemes, for variations in neuronal parameters, and for cell and synapse death. Encoding is shown to be independent of absolute sound level and to vary linearly with interaural intensity difference.

Much information is available on the anatomical organization and neurophysiological properties of the major cell types in the dorsal cochlear nucleus (DCN). The complicated response properties of individual cells and units in the DCN indicate that substantial information processing already occurs at the level of the DCN. A large number of connectional hypotheses have been put forward to explain various aspects of the response characteristics of DCN cells, but many of the consequences of these hypotheses have not been investigated quantitatively. In this paper, we investigate these hypotheses by constructing and testing mathematical and computational models and compare our results to those of previous modeling studies. The simplest versions of our models include auditory nerve (AN) fibers, type II cells (inhibitory interneurons) and type IV cells (fusiform and giant cells). The model response maps, i.e., the pattern of output of model type IV cells, generated by the simplest model have some but not all the features of the experimental response maps of type IV neurons. In particular, the excitatory region which occurs at best frequency is not isolated and the excitatory region at low frequencies and high amplitude is narrower than observed. Since experimental evidence exists that some of the connections between these cell types are divergent or convergent across adjacent isofrequency sheets, the effect of such convergence and divergence was then investigated. Response maps so obtained reproduce many of the qualitative features of the experimental maps. Enlargement of the model by including inhibitory interneurons (e.g., stellate cells) that receive convergent input from AN fibers and which inhibit type II cells results in the generation of response maps that, with some variations in connectional patterns and physiological properties of neurons, match most of the essential features seen in the large variety of experimental maps obtained from the cat DCN.

A number of investigators have published measurements of the outputs of single neurons from the lateral superior olive (LSO) of the cat for a variety of auditory signals. The response curves show a very wide range of shapes and thresholds. In this paper, the single neuron response curves predicted by a previously published model for the encoding of azimuthal location by the LSO to the experimental curves are compared. The predicted curves are in good qualitative agreement with the experiments and, in addition, the model provides an explanation of the seemingly paradoxical drop in output as interaural intensity difference (IID) is held fixed and absolute intensities are raised. The particular shape of the response curve depends on the location of the LSO neuron examined. In the model, two possible developmental programs to form the adult pattern of connections from the anteroventral cochlear nucleus and the medial nucleus of the trapezoid body onto the LSO are also examined. In the first, connections are made by a forward stochastic process in which there is a limit on the numbers of synapses formed on each LSO cell. In the second, there is no such limit, but in later developmental stages pruning of synapses occurs which reduces their number to the limit. Both schemes give similar adult connectional patterns. The LSO response curves generated by the model are similar to those observed in LSO neurons of the developing gerbil by Sanes and Rubel [J. Neurosci. 8, 682–700 (1988)]. Thus the model mechanism not only encodes azimuthal location by activity across the population of neurons, but is also consistent with single unit neurophysiological measurements of LSO output in both developing and adult animals.

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++-dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.

The localization of induced and constitutive acid phosphatase activity in Euglena was studied by light and electron microscopy, using two different cytochemical methods. Cells grown in high phosphate medium have constitutive acid phosphatase activity in three regions: in the Golgi complex, around the paramylum bodies, and in the peri-reservoir vesicles. Cells that have formed an induced acid phosphatase by exposure to a phosphate-deficient medium have, in addition to the constitutive activity localized exactly as in the uninduced cell, a strong activity in the pellicle. The induced activity is not uniformly distributed over the pellicle, but is localized at the notch of each pellicle complex, near a group of about four fibrils and near a characteristic vesicle of the endoplasmic reticulum. In the cytostome, where fission begins during division, there is an alternation of large and small pellicle complexes, both of which have induced phosphatase activity. A similar alternation is seen over the entire pellicle of dividing cells.