Motor Neuron Development Research Articles

Potassium Chloride Cotransporter‐3 (KCC3) in Motor Neurons

The K‐Cl cotransporter KCC3 is an electroneutral membrane transporter of the SLC12A gene family. It uses the K+ gradient established by the Na+/K+ ATPase to efflux K+, Cl−, and obligatory water from cells. KCC3 loss‐of‐function (LOF) mutations have been found to cause the disease Agenesis of the Corpus Callosum with Peripheral Neuropathy (ACCPN). ACCPN patients suffer from intellectual disability, dysmorphic features, hallucinations, degrading ambulation, and a severe, progressive sensorimotor neuropathy. Previous literature has confirmed that both global KCC3 knock‐out mice and neuron‐specific knock‐out (KO) mice recapitulate the severe sensorimotor deficits seen in patients. Furthermore, mice with KCC3 deleted in a subset of sensory neurons (parvalbumin‐positive proprioceptive neurons) also show a locomotor deficit. Thus, KCC3 dysfunction in sensory neurons may be a key to ACCPN pathogenesis; however, it is unknown if KCC3 dysfunction in sensory neurons could account for the entire range of patient symptoms, that is, both sensory and motor neuropathy. From the spinal cord, motor neurons extend their axons to the periphery where they synapse onto skeletal muscles. Reciprocally, sensory neurons, specifically proprioceptive neurons, bring information from muscles back to the spinal cord, where they synapse onto motor neurons and interneurons. Therefore, KCC3 LOF directly in motor neurons, in sensory neurons, or in both types of neurons could be responsible for the ACCPN phenotype.To investigate the participation of motor neurons, we used a mouse line that expresses the CRE recombinase under the control of the HB9 promoter. HB9 is a transcription factor expressed during peak motor neuron development at mouse embryonic days E9.5–10.5. We crossed HB9‐CRE mice with a KCC3‐floxed line to generate mice with KCC3 deleted in motor neurons (HB9‐CRE x KCC3‐flox). We then subjected cohorts of HB9‐positive KCC3‐flox mice versus HB9‐negative KCC3‐flox mice to locomotor tests. The HB9‐positive KCC3‐flox mice did not show any deficits in the accelerated rotarod test, which assesses balance and overall locomotion performance, nor in the elevated balance beam task, which assesses finer movement coordination. Absence of a phenotype indicates that targeting KCC3 deletion in motor neurons does not contribute to the development of neuropathy. These data suggest that the motor phenotype observed in ACCPN patients might be secondary to the sensory deficit. Such an indirect effect is possible as motor neurons need proper sensory feedback from muscles to appropriately coordinate posture and movement.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S12-3. Anti-LRP4 autoantibodies in Japanese patients with myasthenia gravis

Low-density lipoprotein receptor-related protein 4 (LRP4) is crucial membrane protein in the development and function of neuromuscular junctions and motor neurons. And, it is currently known myasthenia gravis (MG) is caused by antibodies to the acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and LRP4. MuSK and LRP4 are coreceptors for agrin in the signaling pathway that causes clustering of AChR. We demonstrated anti-LRP4 antibodies played a key role in MG. LRP4 is essential for maintaining the structural and functional integrity of the neuromuscular junction and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms. MG with anti-LRP4 antibodies (LRP4-MG) is considered a rare subtype of MG, however, LRP4 autoantibodies have been recently detected in some motor neuron disease (MND) patients, and LRP4 is required for presynaptic and postsynaptic differentiation. We developed the luciferase immunoprecipitation systems for LRP4 autoantibodies detection and with this assay we studied for four years the sera from Japanese patients who had been suspected of MG or other neurological diseases with general myasthenia and/or muscle weakness. The clinical and therapeutic implications of the anti-LRP4 antibody positivity remain to be clarified, and further studies are necessary to elucidate the pathogenic potential of these autoantibodies.

Motor Unit Number Estimate and Isometric Hand Grip Strength in Military Veterans with or Without Muscular Complaints: Reference Values for Longitudinal Follow-up.

It remains unclear if Gulf War (GW) veterans have a higher risk of developing motor neuron disorder. We intended to establish baseline neurophysiological values, including thenar motor unit number estimate (MUNE) and isometric hand grip (IHG) strength, to compare future follow-ups of deployed GW veterans with or without muscular complaints. We evaluated 19 GW veterans with self-reported weakness, cramps, or excessive muscle fatigue (Ill-19) and compared them with 18 controls without such muscular complaints (C-18). We performed MUNE on hand thenar muscles using adapted multipoint stimulation method for Ill-19 and 15 controls (C-15). We measured IHG strength (maximum force, endurance, and fatigue level) on Ill-19 and C-18 with a hand dynamometer. We performed nerve conduction studies on all study participants to determine which subjects had mild carpal tunnel syndrome (CTS). We compared the MUNE and IHG strength measures between Ill group and controls and between those with CTS and those without CTS. We obtained thenar MUNE of Ill-19 (95% CI of mean: 143-215; mean age: 46 yr) and compared it with that of C-15 (95% CI of mean: 161-230; mean age: 45 yr), and 95% of CI of mean among IHG strength variables (maximum force: 324-381 Newton; endurance: 32-42 s; fatigue level: 24%-33%) compared with C-18 (maximum force: 349-408 Newton; endurance: 35-46 s; fatigue level: 21%-27%). There was no significant difference in either MUNE or IHG strength between Ill-19 group and controls. The MUNE and IHG maximum forces were significantly lower in those with CTS compared with those without CTS. As a surrogate of mild CTS, the median versus ulnar distal sensory latency on nerve conduction study was only weakly associated with MUNE, maximum force, and fatigue level, respectively. To our knowledge, no published study on MUNE reference values of military veteran population has been available. The quantifiable values of both thenar MUNE and IHG strength of military veterans serve as baselines for our longitudinal follow-up of motor neuron function of deployed troops. These reference values are also useful for other laboratories to study veterans' motor system with or without mild CTS.

Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

Temporal Expression of Mutant TDP-43 Correlates with Early Amyotrophic Lateral Sclerosis Phenotype and Motor Weakness.

Background: Mutant transactive response DNA-binding protein (TDP-43) is closely correlated to the inherited form of amyotrophic lateral sclerosis (ALS). TDP-43 transgenic rats can reproduce the core phenotype of ALS and constitutive expression of TDP-43 caused postnatal death.Objective: The study aimed to understand whether neurologic deficiency caused by mutant TDP-43 is dependent on its temporal expression.Method: Transgenic rats were established that express mutant human TDP-43 (M337V substitution) in neurons, then a Tet-off system was used to regulate its expression.Results: TDP-43 mutant transgenic rats developed significant weakness after the transgene was activated. Rats with expression of mutant TDP-43 at 30 days showed a more aggressive phenotype. More severe pathological changes in neurogenic atrophy were observed in these rats.Conclusion: Temporal expression of mutant TDP-43 in neurons promoted serious phenotype in rats. The dysfunction of TDP-43 had a profound impact on the development of motor neurons and skeletal muscles.

Application of Zebrafish and Knockdown Technology to Define Progranulin Neuronal Function.

The zebrafish (Danio rerio), a small tropical fish, has become a powerful model for the study of early vertebrate development, human diseases, and drug screening. Zebrafish provides large numbers of optically clear embryos, and its development is very rapid. Overexpression or under-expression of proteins can be effectively achieved by microinjection of mRNA or morpholino antisense oligonucleotides (MOs), respectively, into developing embryos at the 1-2 cell stage. The function of a particular protein can be revealed by correlating gene expression patterns with the phenotypes observed from over- or under-expression. We defined the expression pattern of zebrafish progranulin A (zfPGRN-A), an orthologue to the single human PGRN by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). The MO-mediated knockdown of zfPGRN-A expression generated embryos that display abnormal motor neuron development resulting in touch-evoked swimming deficits.

Comparative analysis of the transcriptome responses of zebrafish embryos after exposure to low concentrations of cadmium, cobalt and copper

Metal toxicity is a global environmental challenge. Fish are particularly prone to metal exposure, which can be lethal or cause sublethal physiological impairments. The objective of this study was to investigate how adverse effects of chronic exposure to non-toxic levels of essential and non-essential metals in early life stage zebrafish may be explained by changes in the transcriptome. We therefore studied the effects of three different metals at low concentrations in zebrafish embryos by transcriptomics analysis. The study design compared exposure effects caused by different metals at different developmental stages (pre-hatch and post-hatch). Wild-type embryos were exposed to solutions of low concentrations of copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) until 96h post-fertilization (hpf) and microarray experiments were carried out to determine transcriptome profiles at 48 and 96hpf. We found that the toxic metal cadmium affected the expression of more genes at 96hpf than 48hpf. The opposite effect was observed for the essential metals cobalt and copper, which also showed enrichment of different GO terms. Genes involved in neuromast and motor neuron development were significantly enriched, agreeing with our previous results showing motor neuron and neuromast damage in the embryos. Our data provide evidence that the response of the transcriptome of fish embryos to metal exposure differs for essential and non-essential metals.

Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo

Background: Previous work in the zebrafish embryo has shown that laminin γ-3 ( lamc3) is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.

Neonatal exposure to monosodium glutamate results in dysmorphology of orofacial lower motor neurons

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and is stored and released by both neurons and astrocytes. Despite the important role of glutamate as a neurotransmitter, high levels of extracellular glutamate can result in excitotoxicity and apoptosis. Monosodium glutamate (MSG) is a naturally occurring sodium salt of glutamic acid that is used as a flavour enhancer in many processed foods. Neonatal exposure to MSG has been shown to result in neurodegeneration in several forebrain regions, characterised by neuronal loss and neuroendocrine abnormalities. However, the brainstem effects of neonatal MSG exposure have not been investigated. It is therefore hypothesized that MSG exposure during the early postnatal period would impact brainstem lower motor neurons involved in feeding behaviour. The effect of neonatal MSG exposure on brainstem lower motor neurons was investigated by exposing rat pups to either 4 mg/g MSG or saline from postnatal day (P) 4 through 10. On P28, brains were preserved by vascular perfusion with fixative, frozen sectioned and stained for Nïssl substance. The number, size and shape of brainstem motor neurons were compared between MSG and saline-exposed animals. MSG exposure had no impact on the total number of neurons in the nuclei examined. However, MSG exposure was associated with a significant increase in the number of round somata in both the trigeminal and facial nuclei. Furthermore, MSG exposure resulted in significantly smaller neurons in all motor nuclei examined. These results suggest that neonatal exposure to MSG impacts the development of brainstem lower motor neurons which may impact feeding and swallowing behaviours in young animals.

HuD and the Survival Motor Neuron Protein Interact in Motoneurons and Are Essential for Motoneuron Development, Function, and mRNA Regulation.

Motoneurons establish a critical link between the CNS and muscles. If motoneurons do not develop correctly, they cannot form the required connections, resulting in movement defects or paralysis. Compromised development can also lead to degeneration because the motoneuron is not set up to function properly. Little is known, however, regarding the mechanisms that control vertebrate motoneuron development, particularly the later stages of axon branch and dendrite formation. The motoneuron disease spinal muscular atrophy (SMA) is caused by low levels of the survival motor neuron (SMN) protein leading to defects in vertebrate motoneuron development and synapse formation. Here we show using zebrafish as a model system that SMN interacts with the RNA binding protein (RBP) HuD in motoneurons in vivo during formation of axonal branches and dendrites. To determine the function of HuD in motoneurons, we generated zebrafish HuD mutants and found that they exhibited decreased motor axon branches, dramatically fewer dendrites, and movement defects. These same phenotypes are present in animals expressing low levels of SMN, indicating that both proteins function in motoneuron development. HuD binds and transports mRNAs and one of its target mRNAs, Gap43, is involved in axonal outgrowth. We found that Gap43 was decreased in both HuD and SMN mutants. Importantly, transgenic expression of HuD in motoneurons of SMN mutants rescued the motoneuron defects, the movement defects, and Gap43 mRNA levels. These data support that the interaction between SMN and HuD is critical for motoneuron development and point to a role for RBPs in SMA.SIGNIFICANCE STATEMENT In zebrafish models of the motoneuron disease spinal muscular atrophy (SMA), motor axons fail to form the normal extent of axonal branches and dendrites leading to decreased motor function. SMA is caused by low levels of the survival motor neuron (SMN) protein. We show in motoneurons in vivo that SMN interacts with the RNA binding protein, HuD. Novel mutants reveal that HuD is also necessary for motor axonal branch and dendrite formation. Data also revealed that both SMN and HuD affect levels of an mRNA involved in axonal growth. Moreover, expressing HuD in SMN-deficient motoneurons can rescue the motoneuron development and motor defects caused by low levels of SMN. These data support that SMN:HuD complexes are essential for normal motoneuron development and indicate that mRNA handling is a critical component of SMA.

FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis.

SummaryThe FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS.

Casein kinase II phosphorylation of cyclin F at serine 621 regulates the Lys48-ubiquitylation E3 ligase activity of the SCF(cyclin F) complex.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3–5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al. 2016 Nat. Commun. 7, 11253. (doi:10.1038/ncomms11253)). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF(cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF(cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.

Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo.

Gaskell revisited: new insights into spinal autonomics necessitate a revised motor neuron nomenclature.

Several concepts developed in the nineteenth century have formed the basis of much of our neuroanatomical teaching today. Not all of these were based on solid evidence nor have withstood the test of time. Recent evidence on the evolution and development of the autonomic nervous system, combined with molecular insights into the development and diversification of motor neurons, challenges some of the ideas held for over 100years about the organization of autonomic motor outflow. This review provides an overview of the original ideas and quality of supporting data and contrasts this with a more accurate and in depth insight provided by studies using modern techniques. Several lines of data demonstrate that branchial motor neurons are a distinct motor neuron population within the vertebrate brainstem, from which parasympathetic visceral motor neurons of the brainstem evolved. The lack of an autonomic nervous system in jawless vertebrates implies that spinal visceral motor neurons evolved out of spinal somatic motor neurons. Consistent with the evolutionary origin of brainstem parasympathetic motor neurons out of branchial motor neurons and spinal sympathetic motor neurons out of spinal motor neurons is the recent revision of the organization of the autonomic nervous system into a cranial parasympathetic and a spinal sympathetic division (e.g., there is no sacral parasympathetic division). We propose a new nomenclature that takes all of these new insights into account and avoids the conceptual misunderstandings and incorrect interpretation of limited and technically inferior data inherent in the old nomenclature.

Insm1a Regulates Motor Neuron Development in Zebrafish.

Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805. Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

Interactions between LHX3- and ISL1-family LIM-homeodomain transcription factors are conserved in Caenorhabditis elegans

LIM-Homeodomain (LIM-HD) transcription factors are highly conserved in animals where they are thought to act in a transcriptional ‘LIM code’ that specifies cell types, particularly in the central nervous system. In chick and mammals the interaction between two LIM-HD proteins, LHX3 and Islet1 (ISL1), is essential for the development of motor neurons. Using yeast two-hybrid analysis we showed that the Caenorhabditis elegans orthologs of LHX3 and ISL1, CEH-14 and LIM-7 can physically interact. Structural characterisation of a complex comprising the LIM domains from CEH-14 and a LIM-interaction domain from LIM-7 showed that these nematode proteins assemble to form a structure that closely resembles that of their vertebrate counterparts. However, mutagenic analysis across the interface indicates some differences in the mechanisms of binding. We also demonstrate, using fluorescent reporter constructs, that the two C. elegans proteins are co-expressed in a small subset of neurons. These data show that the propensity for LHX3 and Islet proteins to interact is conserved from C. elegans to mammals, raising the possibility that orthologous cell specific LIM-HD-containing transcription factor complexes play similar roles in the development of neuronal cells across diverse species.

JoVE Monthly Highlights: June 2017

Here's a look at what's coming up in the June 2017 issue of JoVE: The World's Premier Video Journal A handy method to test the efficacy of sanitizing techniques is our first feature this month, from JoVE Immunology and Infection. Here, our Authors first spike the gloved hands of volunteers with a test organism, and then employ a hand-washing technique of interest. Then, the remaining organisms are collected, and unwashed and test samples are compared. These results allow researchers to provide recommendations for best cleanliness practices during emerging infectious disease outbreaks. We can all hi-five with confidence to that! As spring is in full bloom so are allergies, and these are the subjects of our next video highlight. In JoVE Medicine, our Authors describe a novel diagnostic approach for determining specific types of grass pollen allergy using a cohort of patients from Southern China - an area with previously limited data on local allergens. Here, the researchers employed component-resolved diagnostics involving recombinant or purified allergens, to detect sensitization to specific types of grass pollen in test and control individuals. This allows for more targeted treatment of allergic airway diseases in patients with allergic rhinitis or asthma, and that's nothing to sneeze at! From grassy gametes to adult plant cells, next we showcase a method for visualizing plant cell wall components, from JoVE Biochemistry. Here, our Authors demonstrate a technique for accurately distinguishing and quantifying cell wall constituents - including those with similar chemical structures such as lignin, cellulose and hemicellulose - using a combination of Raman imaging and multivariate analysis. This method overcomes the barriers to studies of dynamic changes in cell wall components during chemical, physical or biological treatments. Finally this month we move from the sessile to the mobile and examine motor neuron development with JoVE Neuroscience. Studying late stage-16 Drosophila embryos, our Author details an immunohistochemistry method to analyze axonal motor neuron projections. By positioning fixed embryos in a filleted arrangement and staining with FasII antibody, the resulting preparations are ideal for the characterization of genes involved in motor axon path-finding and target recognition. It's a neat little technique, with lots of muscle. You've just had a sneak peek of the June 2017 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The World's Premier Video Journal.

MotomiRs: miRNAs in Motor Neuron Function and Disease.

MiRNAs are key regulators of the mammalian transcriptome that have been increasingly linked to degenerative diseases of the motor neurons. Although many of the miRNAs currently incriminated as participants in the pathogenesis of these diseases are also important to the normal development and function of motor neurons, at present there is no knowledge of the complete miRNA profile of motor neurons. In this review, we examine the current understanding with respect to miRNAs that are specifically required for motor neuron development, function and viability, and provide evidence that these should be considered as a functional network of miRNAs which we have collectively termed MotomiRs. We will also summarize those MotomiRs currently known to be associated with both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), and discuss their potential use as biomarkers.

Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice

De novo heterozygous missense mutations in the gene encoding translation elongation factor eEF1A2 have recently been found to give rise to neurodevelopmental disorders. Children with mutations in this gene have developmental delay, epilepsy, intellectual disability and often autism; the most frequently occurring mutation is G70S. It has been known for many years that complete loss of eEF1A2 in mice causes motor neuron degeneration and early death; on the other hand heterozygous null mice are apparently normal. We have used CRISPR/Cas9 gene editing in the mouse to mutate the gene encoding eEF1A2, obtaining a high frequency of biallelic mutations. Whilst many of the resulting founder (F0) mice developed motor neuron degeneration, others displayed phenotypes consistent with a severe neurodevelopmental disorder, including sudden unexplained deaths and audiogenic seizures. The presence of G70S protein was not sufficient to protect mice from neurodegeneration in G70S/− mice, showing that the mutant protein is essentially non-functional.

The assembly of developing motor neurons depends on an interplay between spontaneous activity, type II cadherins and gap junctions.

A core structural and functional motif of the vertebrate central nervous system is discrete clusters of neurons or 'nuclei'. Yet the developmental mechanisms underlying this fundamental mode of organisation are largely unknown. We have previously shown that the assembly of motor neurons into nuclei depends on cadherin-mediated adhesion. Here, we demonstrate that the emergence of mature topography among motor nuclei involves a novel interplay between spontaneous activity, cadherin expression and gap junction communication. We report that nuclei display spontaneous calcium transients, and that changes in the activity patterns coincide with the course of nucleogenesis. We also find that these activity patterns are disrupted by manipulating cadherin or gap junction expression. Furthermore, inhibition of activity disrupts nucleogenesis, suggesting that activity feeds back to maintain integrity among motor neurons within a nucleus. Our study suggests that a network of interactions between cadherins, gap junctions and spontaneous activity governs neuron assembly, presaging circuit formation.