Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

Background: Cytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. Results: Here we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. Conclusions: The zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

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LanCL1 promotes motor neuron survival and extends the lifespan of amyotrophic lateral sclerosis mice

Cell Death and Differentiation ◽

10.1038/s41418-019-0422-6 ◽

2019 ◽

Vol 27 (4) ◽

pp. 1369-1382 ◽

Cited By ~ 4

Author(s):

Honglin Tan ◽

Mina Chen ◽

Dejiang Pang ◽

Xiaoqiang Xia ◽

Chongyangzi Du ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

Neuronal Survival ◽

Disease Onset ◽

Alternative Mechanism ◽

Specific Expression ◽

Motor Neuron Survival ◽

Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Improving neuronal survival in ALS remains a significant challenge. Previously, we identified Lanthionine synthetase C-like protein 1 (LanCL1) as a neuronal antioxidant defense gene, the genetic deletion of which causes apoptotic neurodegeneration in the brain. Here, we report in vivo data using the transgenic SOD1G93A mouse model of ALS indicating that CNS-specific expression of LanCL1 transgene extends lifespan, delays disease onset, decelerates symptomatic progression, and improves motor performance of SOD1G93A mice. Conversely, CNS-specific deletion of LanCL1 leads to neurodegenerative phenotypes, including motor neuron loss, neuroinflammation, and oxidative damage. Analysis reveals that LanCL1 is a positive regulator of AKT activity, and LanCL1 overexpression restores the impaired AKT activity in ALS model mice. These findings indicate that LanCL1 regulates neuronal survival through an alternative mechanism, and suggest a new therapeutic target in ALS.

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Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

10.1101/2021.10.11.464011 ◽

2021 ◽

Author(s):

Jin Hao ◽

Michael F Wells ◽

Gengle Niu ◽

Irune Guerra San Juan ◽

Francesco Limone ◽

...

Keyword(s):

Motor Neuron ◽

Neurodegenerative Disease ◽

Motor Neurons ◽

Inhibition Mechanism ◽

Loss Of Function ◽

Neurodegenerative Process ◽

Lysosomal Dysfunction ◽

Human Motor ◽

Lateral Sclerosis ◽

Lysosomal Acidification

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

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The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification

Development ◽

10.1242/dev.127.19.4239 ◽

2000 ◽

Vol 127 (19) ◽

pp. 4239-4252 ◽

Cited By ~ 2

Author(s):

S. Hallam ◽

E. Singer ◽

D. Waring ◽

Y. Jin

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Expression Patterns ◽

Loss Of Function ◽

Variable Expression ◽

C Elegans ◽

Helix Loop Helix ◽

Ventral Cord ◽

Bhlh Proteins ◽

Neuronal Type

The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. Here we report the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family.

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AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/−SMA model mice

Human Molecular Genetics ◽

10.1093/hmg/ddz142 ◽

2019 ◽

Vol 28 (19) ◽

pp. 3199-3210 ◽

Cited By ~ 9

Author(s):

Kevin A Kaifer ◽

Eric Villalón ◽

Benjamin S O'Brien ◽

Samantha L Sison ◽

Caley E Smith ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Molecular Mechanisms ◽

Viral Vector ◽

Muscular Atrophy ◽

Induced Pluripotent Stem Cell ◽

Survival Motor Neuron ◽

Virus Serotype

Abstract Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/− SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

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Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo

Human Molecular Genetics ◽

10.1093/hmg/ddq203 ◽

2010 ◽

Vol 19 (15) ◽

pp. 3102-3102 ◽

Cited By ~ 3

Author(s):

E. Kabashi ◽

L. Lin ◽

M. L. Tradewell ◽

P. A. Dion ◽

V. Bercier ◽

...

Keyword(s):

Motor Deficits ◽

Loss Of Function ◽

Gain And Loss

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An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

10.1101/2020.11.01.362269 ◽

2020 ◽

Author(s):

◽

Loren Ornelas ◽

Emilda Gomez ◽

Lindsay Panther ◽

Aaron Frank ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Rna Splicing ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Data Independent Acquisition ◽

Induced Pluripotent ◽

Als Patients ◽

And Control

SummaryNeurodegenerative diseases present a challenge for systems biology, due to the lack of reliable animal models and the difficulties in obtaining samples from patients at early stages of disease, when interventions might be most effective. Studying induced pluripotent stem cell (iPSC)-derived neurons could overcome these challenges and dramatically accelerate and broaden therapeutic strategies. Here we undertook a network-based multi-omic characterization of iPSC-derived motor neurons from ALS patients carrying genetically dominant hexanucleotide expansions in C9orf72 to gain a deeper understanding of the relationship between DNA, RNA, epigenetics and protein in the same pool of tissue. ALS motor neurons showed the expected C9orf72-related alterations to specific nucleoporins and production of dipeptide repeats. RNA-seq, ATAC-seq and data-independent acquisition mass-spectrometry (DIA-MS) proteomics were then performed on the same motor neuron cultures. Using integrative computational methods that combined all of the omics, we discovered a number of novel dysregulated pathways including biological adhesion and extracellular matrix organization and disruption in other expected pathways such as RNA splicing and nuclear transport. We tested the relevance of these pathways in vivo in a C9orf72 Drosophila model, analyzing the data to determine which pathways were causing disease phenotypes and which were compensatory. We also confirmed that some pathways are altered in late-stage neurodegeneration by analyzing human postmortem C9 cervical spine data. To validate that these key pathways were integral to the C9 signature, we prepared a separate set of C9orf72 and control motor neuron cultures using a different differentiation protocol and applied the same methods. As expected, there were major overall differences between the differentiation protocols, especially at the level of in individual omics data. However, a number of the core dysregulated pathways remained significant using the integrated multiomic analysis. This new method of analyzing patient specific neural cultures allows the generation of disease-related hypotheses with a small number of patient lines which can be tested in larger cohorts of patients.

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“Stretch-Growth” of Motor Axons in Custom Mechanobioreactors to Generate Long-Projecting Axonal and Axonal-Myocyte Constructs

10.1101/598755 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kritika S. Katiyar ◽

Laura A. Struzyna ◽

Suradip Das ◽

D. Kacy Cullen

Keyword(s):

Spinal Cord ◽

Nervous System ◽

Motor Neuron ◽

Motor Neurons ◽

Motor Axon ◽

Central Feature ◽

Motor Axons ◽

Ganglion Neurons

AbstractThe central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to an often-distant target muscle. However, to date in vitro models have not replicated this central feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of micro-tissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from the spinal cords of rats and induced to form engineered micro-spheres via forced aggregation in custom micro-wells. This three-dimensional micro-tissue yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were systematically optimized to generate parameters for robust and efficient “stretch-growth” of motor axons. We found that axons projecting from motor neuron aggregates were able to respond to axon displacement rates at least 10 times greater than that tolerated by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared to benchmark stretch-grown axons from sensory dorsal root ganglion neurons, revealing similar axon densities yet increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and then stretch-grown to create novel long-projecting axonal-myocyte constructs that better recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal cord motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue engineered “living scaffolds” for targeted axon tract reconstruction following nervous system injury or disease.Significance StatementWe have developed novel axon tracts of unprecedented lengths spanning either two discrete populations of neurons or a population of neurons and skeletal myocytes. This is the first demonstration of “stretch-grown” motor axons that recapitulate the structure of spinal motor neurons in vivo by projecting long axons from a pool of motor neurons to distant targets, and may have applications as anatomically inspired in vitro test beds to study mechanisms of axon growth, development, and neuromuscular function in anatomically accurate axo-myo constructs; as well as serve as “living scaffolds” in vivo for targeted axon tract reconstruction following nervous system trauma.

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Functional characterization of Neurofilament Light b splicing and misbalance in zebrafish

10.1101/2020.04.27.064063 ◽

2020 ◽

Author(s):

DL Demy ◽

ML Campanari ◽

R Munoz-Ruiz ◽

HD Durham ◽

BJ Gentil ◽

...

Keyword(s):

Motor Neurons ◽

Neuronal Development ◽

Rna Binding ◽

De Novo ◽

Functional Characterization ◽

Axon Growth ◽

Motor Deficits ◽

Neurofilament Light ◽

Cytoplasmic Inclusions

AbstractNeurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in their differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit, NEFL, which expression is reduced in motor neurons in Amyotrophic Lateral Sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects NF assembly and motor axon growth, with resulting motor deficits. This equilibrium is also disrupted upon partial depletion of TDP-43, a RNA binding protein that is mislocalized into cytoplasmic inclusions in ALS. The study supports interaction of NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

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Modeling motor neuron resilience in ALS using stem cells

10.1101/399659 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ilary Allodi ◽

Jik Nijssen ◽

Julio Aguila Benitez ◽

Christoph Schweingruber ◽

Andrea Fuchs ◽

...

Keyword(s):

Stem Cells ◽

Motor Neuron ◽

Motor Neurons ◽

Embryonic Stem ◽

Spinal Motor Neurons ◽

Neuronal Progenitors ◽

Oculomotor Neurons ◽

Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

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Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b

Development ◽

10.1242/dev.127.7.1349 ◽

2000 ◽

Vol 127 (7) ◽

pp. 1349-1358 ◽

Cited By ~ 10

Author(s):

A. Pattyn ◽

M. Hirsch ◽

C. Goridis ◽

J.F. Brunet

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Early Stage ◽

Control Point ◽

Homeobox Gene ◽

Homeodomain Protein ◽

Loss Of Function ◽

Dorsoventral Patterning ◽

Cns Neurons ◽

Mantle Layer

Motor neurons are a widely studied model of vertebrate neurogenesis. They can be subdivided in somatic, branchial and visceral motor neurons. Recent studies on the dorsoventral patterning of the rhombencephalon have implicated the homeobox genes Pax6 and Nkx2.2 in the early divergence of the transcriptional programme of hindbrain somatic and visceral motor neuronal differentiation. We provide genetic evidence that the paired-like homeodomain protein Phox2b is required for the formation of all branchial and visceral, but not somatic, motor neurons in the hindbrain. In mice lacking Phox2b, both the generic and subtype-specific programs of motoneuronal differentiation are disrupted at an early stage. Most motor neuron precursors die inside the neuroepithelium while those that emigrate to the mantle layer fail to switch on early postmitotic markers and to downregulate neuroepithelial markers. Thus, the loss of function of Phox2b in hindbrain motor neurons exemplifies a novel control point in the generation of CNS neurons.

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