Mutant Motoneurons Research Articles

Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1564-y) contains supplementary material, which is available to authorized users.

Dscam1 Is Required for Normal Dendrite Growth and Branching But Not for Dendritic Spacing inDrosophilaMotoneurons

Down syndrome cell adhesion molecule, Dscam, serves diverse neurodevelopmental functions, including axon guidance and synaptic adhesion, as well as self-recognition and self-avoidance, depending on the neuron type, brain region, or species under investigation. In Drosophila, the extensive molecular diversity that results from alternative splicing of Dscam1 into >38,000 isoforms provides neurons with a unique molecular code for self-recognition in the nervous system. Each neuron produces only a small subset of Dscam1 isoforms, and distinct Dscam1 isoforms mediate homophilic interactions, which in turn, result in repulsion and even spacing of self-processes, while allowing contact with neighboring cells. While these mechanisms have been shown to underlie mushroom body development and spacing of mechanosensory neuron dendrites, here we report that Dscam1 plays no role in adult Drosophila motoneuron dendrite spacing, but is required for motoneuron dendritic growth. Targeted expression of Dscam-RNAi in an identified flight motoneuron did not impact dendrite spacing, but instead produced overgrowth. Increasing the knockdown strength severely reduced dendritic growth and branching. Similarly, Dscam mutant motoneurons in an otherwise control background (MARCM) were completely devoid of mature dendrites. These data suggest that Dscam1 is required cell autonomously for normal adult motoneuron dendrite growth in Drosophila. This demonstrates a previously unreported role of Drosophila Dscam1 in central neuron development, and expands the current understanding that Dscam1 operates as a cell adhesion molecule that mediates homophilic repulsion.

Mechanisms for axon maintenance and plasticity in motoneurons: alterations in motoneuron disease

In motoneuron disease and other neurodegenerative disorders, the loss of synapses and axon branches occurs early but is compensated by sprouting of neighboring axon terminals. Defective local axonal signaling for maintenance and dynamics of the axonal microtubule and actin cytoskeleton plays a central role in this context. The molecular mechanisms that lead to defective cytoskeleton architecture in two mouse models of motoneuron disease are summarized and discussed in this manuscript. In the progressive motor neuropathy (pmn) mouse model of motoneuron disease that is caused by a mutation in the tubulin-specific chaperone E gene, death of motoneuron cell bodies appears as a consequence of axonal degeneration. Treatment with bcl-2 overexpression or with glial-derived neurotrophic factor prevents loss of motoneuron cell bodies but does not influence the course of disease. In contrast, treatment with ciliary neurotrophic factor (CNTF) significantly delays disease onset and prolongs survival of pmn mice. This difference is due to the activation of Stat-3 via the CNTF receptor complex in axons of pmn mutant motoneurons. Most of the activated Stat-3 protein is not transported to the nucleus to activate transcription, but interacts locally in axons with stathmin, a protein that destabilizes microtubules. This interaction plays a major role in CNTF signaling for microtubule dynamics in axons. In Smn-deficient mice, a model of spinal muscular atrophy, defects in axonal translocation of β-actin mRNA and possibly other mRNA species have been observed. Moreover, the regulation of local protein synthesis in response to signals from neurotrophic factors and extracellular matrix proteins is altered in motoneurons from this model of motoneuron disease. These findings indicate that local signals are important for maintenance and plasticity of axonal branches and neuromuscular endplates, and that disturbances in these signaling mechanisms could contribute to the pathophysiology of motoneuron diseases.

Candidate ALS Therapeutics Motor toward “In Vitro Clinical Trials”

Conducting drug discovery efforts in patient- and disease-specific cells can maximize their likelihood of success. In this issue of Cell Stem Cell, Yang et al. (2013) demonstrate the power of lineage-specific cell-based drug screens by identifying a compound that promotes survival of stem-cell-derived ALS mutant motor neurons.

The extracellular matrix proteoglycan perlecan facilitates transmembrane semaphorin-mediated repulsive guidance

The Drosophila transmembrane semaphorin-1a (Sema-1a) is a repulsive guidance cue that uses the Plexin A (PlexA) receptor during neural development. Sema-1a is required in axons to facilitate motor axon defasciculation at guidance choice points. We found that mutations in the trol gene strongly suppress Sema-1a-mediated repulsive axon guidance. trol encodes the phylogenetically conserved secreted heparan sulfate proteoglycan (HSPG) perlecan, a component of the extracellular matrix. Motor axon guidance defects in perlecan mutants resemble those observed in Sema-1a- and PlexA-null mutant embryos, and perlecan mutants genetically interact with PlexA and Sema-1a. Perlecan protein is found in both the CNS and the periphery, with higher expression levels in close proximity to motor axon trajectories and pathway choice points. Restoring perlecan to mutant motor neurons rescues perlecan axon guidance defects. Perlecan augments the reduction in phospho-focal adhesion kinase (phospho-FAK) levels that result from treating insect cells in vitro with Sema-1a, and genetic interactions among integrin, Sema-1a, and FAK in vivo support an antagonistic relationship between Sema-1a and integrin signaling. Therefore, perlecan is required for Sema-1a-PlexA-mediated repulsive guidance, revealing roles for extracellular matrix proteoglycans in modulating transmembrane guidance cue signaling during neural development.

In vivo imaging reveals rapid morphological reactions of astrocytes towards focal lesions in an ALS mouse model

Pathophysiology of the motoneuron disease amyotrophic lateral sclerosis (ALS) is non-cell-autonomous. In mouse models of familiar ALS, neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring glial cells. In vivo imaging by two-photon laser-scanning microscopy was used to study rapid morphological reactions of astroglial cells towards laser-induced axonal transection in ALS-linked transgenic SOD1G93A mice. In the affected lateral spinal cord, mutated astroglial cells extended branches towards injured axons within a time frame of minutes to hours post lesion while in control animals astrocytes lack any rapid morphological alteration within the studied time frame. This suggests that astrocytes partially contribute to the rapid response of non-neuronal cells to acute axonal lesions in ALS mice.

Loss of ATF2 Function Leads to Cranial Motoneuron Degeneration during Embryonic Mouse Development

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.

In Vivo Imaging Reveals Distinct Inflammatory Activity of CNS Microglia versus PNS Macrophages in a Mouse Model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1G93A mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ∼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.

The Effect of Training on Motoneuron Survival in Amyotrophic Lateral Sclerosis: Which Motoneuron Type is Saved?

The Effect of Training on Motoneuron Survival in Amyotrophic Lateral Sclerosis: Which Motoneuron Type is Saved?

Early Excitability Changes in Lumbar Motoneurons of Transgenic SOD1G85Rand SOD1G93A-LowMice

This work characterizes the properties of wild-type (WT) mouse motoneurons in the second postnatal week and compares these at the same age and in the same conditions to those of two different SOD1 mutant lines used as models of human amyotrophic lateral sclerosis (ALS), the SOD1(G93A) low expressor line and SOD1(G85R) line, to describe any changes in the functional properties of mutant motoneurons (Mns) that may be related to the pathogenesis of human ALS. We show that very early changes in excitability occur in SOD1 mutant Mns that have different properties from those of WT animals. The SOD1(G93A-Low) low expressor line displays specific differences that are not found in other mutant lines including a more depolarized membrane potential, larger spike width, and slower spike rise slope. With current pulses SOD1(G93A-Low) were hyperexcitable, but both mutants had a lower gain with current ramps stimulation. Changes in the threshold and intensities of Na(+) and Ca(2+) persistent inward currents were also observed. Low expressor mutants show reduced total persistant inward currents compared with WT motoneurons in the same recording conditions and give arguments toward modifications of the balance between Na(+) and Ca(2+) persistent inward currents. During the second week postnatal, SOD1(G93A-Low) lumbar motoneurons appear more immature than those of SOD1(G85R) compared with WT and we propose that different time course of the disease, possibly linked with different toxic properties of the mutated protein in each model, may explain the discrepancies between excitability changes described in the different models.

Valproic acid blocks excitability in SMA type I mouse motor neurons

Valproic acid (VPA), an antiepileptic drug and HDAC inhibitor, has been identified as a drug candidate for spinal muscular atrophy (SMA), a motoneuron disorder for which currently no effective therapy is available. Based on its potential to up-regulate SMN expression from the SMN2 gene in fibroblasts and lymphoblastoid cell lines from SMA patients, we analysed the effects of VPA in isolated motoneurons from Smn −/− ;SMN2 mice, a model for SMA type I. Treatment with VPA increased Smn expression but unexpectedly also led to reduced growth cone size and reduced excitability in axon terminals of mutant motoneurons. Analysis of Ca 2+ currents and distribution of voltage-gated Ca 2+ channels revealed an inhibitory function of VPA on voltage-gated Ca 2+ channels and possibly also other ion channels that contribute to presynaptic excitability of motoneurons. Our data indicate effects of VPA which might aggravate disease-specific symptoms in SMA patients.

Non-Cell-Autonomous Effect of Human SOD1 G37R Astrocytes on Motor Neurons Derived from Human Embryonic Stem Cells

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the non-cell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mutated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce superoxide and that effect can be reversed by antioxidants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1-mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astrocyte-based cell model.

Changes in the expression of P2X1 and P2X2 purinergic receptors in facial motoneurons after nerve lesions in rodents and correlation with motoneuron degeneration

Involvement of P2X1 and P2X2 purinergic receptors in motoneuron response to injury was investigated with Western blotting and immunohistochemistry and correlated with motoneuron loss, Bcl-2 expression, nitric oxide synthase induction and glial activation. P2X1 was highly induced in rat facial motoneurons after nerve resection, which causes slowly occurring neurodegeneration. P2X1 induction was lower and less persistent after nerve crush, permissive for fiber regeneration. P2X2 expression was found in nuclei of rat facial motoneurons, with nuclear export in the cytoplasm after nerve resection. P2X1 induction in axotomized facial motoneurons was impaired in superoxide dismutase (SOD)1-G93A-mutant mice, a model of motoneuron disease. The data in rats point to a correlation of P2X1 induction with motoneuron degeneration, which also involves P2X2 intracellular changes, rather than with axon regeneration effort. The data in mice show that the SOD1 mutation interferes with injury-elicited P2X1 induction, suggesting alterations of ATP release from mutant motoneurons after damage.

Role of GSK‐3β activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene

Point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase-3 (GSK-3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimer's disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol-3-kinase (PI3-K)/Akt and GSK-3 pathway as well as the caspase-3 pathway, VSC4.1 motoneuron cells were transfected with G93A- or A4V-mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24 h after neuronal differentiation, their viability and intracellular signals, including PI3-K/Akt, GSK-3, heat shock transcription factor-1 (HSTF-1), cytochrome c, caspase-3 and poly(ADP-ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK-3beta-mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK-3beta inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK-3beta inhibitor increased their viability by activating HSTF-1 and by reducing cytochrome c release, caspase-3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3-K/Akt and GSK-3beta. These results suggest that the G93A or A4V mutations inhibit PI3-K/Akt and activate GSK-3beta and caspase-3, thus becoming vulnerable to oxidative stress, and that the GSK-3beta-mediated cell death mechanism is important in G93A and A4V cell death.

Retinal oxidation, apoptosis and age- and sex-differences in the mnd mutant mouse, a model of neuronal ceroid lipofuscinosis

Retinal degeneration is an early and progressive event in many forms of neuronal ceroid lipofuscinoses (NCLs), a heterogeneous group of neurodegenerative disorders with unknown pathogenesis. We here used the mutant motor neuron degeneration (mnd) mouse, a late-infantile NCL variant, to investigate the retinal oxidative state and apoptotic cell death as a function of age and sex. Total superoxide dismutase (SOD) activities and thiobarbituric acid-reactive substance (TBARS) levels revealed progressive increases in retinal oxyradicals and lipid peroxides of mnd mice of both sexes. Female mnd retinas showed a higher oxidation rate and consistently exhibited the 4-hydroxy-2-nonenal (4-HNE)-adducts staining and advanced histopathologic profile when compared to male mnd retinas matched for age. In situ DNA fragmentation (TUNEL staining) appeared in the outer nuclear layer (ONL) as early as 1 month of age. At 4 months, there were more intense and numerous TUNEL-positive cells in the same layer and in the inner nuclear (INL) and ganglion cell (GCL) layers; whereas at 8 months TUNEL staining was restricted to a few scattered cells in the INL and GCL, when a severe retinal cell loss had occurred. Caspase-3 activation confirmed apoptotic demise and its processing turned out to be higher in mnd females than males. These results demonstrate the involvement of oxidation and apoptotic processes in mnd mouse retinopathy and highlight sex-related differences in retinal vulnerability to oxidative stress and damage.

The Neuroprotective Factor Wld<SUP>s</SUP> Does Not Attenuate Mutant SOD1-Mediated Motor Neuron Disease

Selective degeneration and death of motor neurons in SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS) is accompanied by axonal disorganization and reduced slow axonal transport in the three most frequently used mouse models of mutant SOD1-mediated ALS. To test whether suppression of axonal degeneration (frequently known as Wallerian degeneration) could slow disease development, we took advantage of a spontaneous mouse mutant Wld(s) (Wallerian degeneration slow) in which the programmed axonal degenerative process that is normally activated after axonal injury is significantly delayed. Despite its effectiveness in delaying axonal loss in other neurodegenerative models, the presence of Wld(s) did not slow disease onset, ameliorate mutant motor neuron death, axonal degeneration, or preserve synaptic attachments in mice that develop disease from ALS-linked SOD1 mutants SOD1G37R or SOD1G85R. However, presynaptic endings in both the presence and absence of Wld(s) showed high accumulations of mitochondria and synaptic vesicles, implicating errors of retrograde transport as a consequence of SOD1-mutant damage to axons.

Foxo3a induces motoneuron death through the Fas pathway in cooperation with JNK

BackgroundProgrammed cell death of motoneurons in the developing spinal cord is thought to be regulated through the availability of target-derived neurotrophic factors. When deprived of trophic support, embryonic spinal motoneurons in vitro over-express FasL, a ligand activating a Fas-mediated death pathway. How trophic factors regulate the expression of FasL is presently unclear, but two regulators of FasL, FOXO3a (FKHRL1) and JNK have been described to play a role in other cell types. Thus, their potential function in motoneurons was investigated in this study.ResultsWe show here that as a result of removal of neurotrophic factors and the consequent reduction in signalling through the PI3K/Akt pathway, Foxo3a translocates from the cytoplasm to the nucleus where it triggers cell death. Death is reduced in Fas and FasL mutant motoneurons and in the presence of JNK inhibitors indicating that a significant part of it requires activation of the Fas/FasL pathway through JNK.ConclusionsTherefore, in motoneurons as in other cell types, FOXO transcriptional regulators provide an important link between other signalling pathways and the cell death machinery.

Motoneuron Survival Is Enhanced in the Absence of Neuromuscular Junction Formation in Embryos

Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.

GFRα1 Is Required for Development of Distinct Subpopulations of Motoneuron

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a glycosyl-phosphatidylinositol-linked co-receptor, either GFRalpha1 or GFRalpha2. By whole-mount in situ hybridization on embryonic mouse spinal cord, we demonstrate that whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complex and distinct patterns of expression. Most motoneurons purified from Gfra1 null mutant mice had lost their responsiveness to both GDNF and NTN. However, a minority of them ( approximately 25%) retained their ability to respond to both factors, perhaps because they express GFRalpha2. Surprisingly, Gfra2(-/-) motoneurons showed normal survival responses to both GDNF and NTN. Thus, GFRalpha1, but not GFRalpha2, is absolutely required for the survival response of a majority of motoneurons to both GDNF and NTN. In accordance with the phenotype of the mutant motoneurons observed in culture we found the loss of distinct groups of motoneurons, identified by several markers, in the Gfra1(-/-) spinal cords but no gross defects in the Gfra2(-/-) mutant. During their natural programmed cell death period, motoneurons in the Gfra1(-/-) mutant mice undertook increased apoptosis. Taken together these findings support the existence of subpopulations of motoneuron with different trophic requirements, some of them being dependent on the GDNF family.

An Early-Onset Congenic Strain of themotor neuron degeneration (mnd)Mouse

The mouse mutantmotor neuron degeneration (mnd/mnd)has been proposed as a model of neuronal ceroid lipofuscinosis (NCL) on the basis of widespread abnormal accumulating lipopigment and neuronal and retinal degeneration. Clinically, the mutant on a C57Bl/6 genetic background shows a progressive motor abnormality starting by 6 months of age, with death prior to 12 months. Whenmndis outcrossed to the AKR/J genetic background, ca. 40% of themnd/mndF2 progeny show early onset (onset by 4.5–5 months and death by 7 months). A congenic strain ofmndhas now been produced by eight generations of backcross onto the AKR background. Mice on this background show average onset at 4 months, and most are moribund prior to 5.5 months. The early onset appears to correlate with levels of abnormal accumulating material, and should prove useful in elucidating NCL neurodegenerative mechanisms.