Astrocytic expression of ALS-causative mutant FUS leads to TNFa-dependent neurodegeneration in vivo

Genetic mutations that cause Amyotrophic Lateral Sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein Fused in Sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that mtFUS expression causes TNFα upregulation, motor function deficits, and spinal motor neuron loss. We further demonstrate a lack of phenotype in TNFα knockout animals expressing mtFUS, and prevention of neurodegeneration in mtFUS-transduced animals through administration of TNFα neutralizing antibodies. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS, establish that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo, and provide insights identifying FUS-ALS specific potential therapeutic targets.

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FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Science Advances ◽

10.1126/sciadv.abf8660 ◽

2021 ◽

Vol 7 (30) ◽

pp. eabf8660

Author(s):

Nicol Birsa ◽

Agnieszka M. Ule ◽

Maria Giovanna Garone ◽

Brian Tsang ◽

Francesca Mattedi ◽

...

Keyword(s):

Phase Separation ◽

Motor Neurons ◽

Rna Binding ◽

Fragile X ◽

Protein Translation ◽

Fused In Sarcoma ◽

Mental Retardation Protein ◽

Lateral Sclerosis

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

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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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“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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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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Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809609115 ◽

2018 ◽

Vol 115 (47) ◽

pp. E11061-E11070 ◽

Cited By ~ 6

Author(s):

Kyu-Hyeon Yeom ◽

Simon Mitchell ◽

Anthony J. Linares ◽

Sika Zheng ◽

Chia-Ho Lin ◽

...

Keyword(s):

Neuronal Differentiation ◽

Rna Binding ◽

Binding Protein ◽

Embryonic Stem ◽

Specific Expression ◽

Stem Loop ◽

Polypyrimidine Tract Binding Protein ◽

Precursor Rna

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem–loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

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A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

The Journal of Cell Biology ◽

10.1083/jcb.200511068 ◽

2006 ◽

Vol 172 (5) ◽

pp. 733-745 ◽

Cited By ~ 126

Author(s):

Jennifer R. Levy ◽

Charlotte J. Sumner ◽

Juliane P. Caviston ◽

Mariko K. Tokito ◽

Srikanth Ranganathan ◽

...

Keyword(s):

Cell Death ◽

Motor Neuron ◽

Motor Neurons ◽

Mitotic Spindle ◽

Cytoplasmic Dynein ◽

Aggregate Formation ◽

Microtubule Motor ◽

Delayed Recovery

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

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Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons

Biomolecules ◽

10.3390/biom11121898 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1898

Author(s):

Cristina Pinto ◽

Viviana Pérez ◽

Jessica Mella ◽

Miguel Albistur ◽

Teresa Caprile ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Nerve Terminal ◽

Motor Nerve ◽

Motor Nerve Terminal ◽

In Ovo ◽

Wnt Ligands ◽

Postsynaptic Differentiation

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

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Histamine Is an Inducer of the Heat Shock Response in SOD1-G93A Models of ALS

International Journal of Molecular Sciences ◽

10.3390/ijms20153793 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3793 ◽

Cited By ~ 2

Author(s):

Savina Apolloni ◽

Francesca Caputi ◽

Annabella Pignataro ◽

Susanna Amadio ◽

Paola Fabbrizio ◽

...

Keyword(s):

Heat Shock ◽

Motor Neuron ◽

Motor Neurons ◽

Heat Shock Response ◽

Type I ◽

Spine Density ◽

Shock Response ◽

Anti Inflammatory

(1) Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial non-cell autonomous disease where activation of microglia and astrocytes largely contributes to motor neurons death. Heat shock proteins have been demonstrated to promote neuronal survival and exert a strong anti-inflammatory action in glia. Having previously shown that the pharmacological increase of the histamine content in the central nervous system (CNS) of SOD1-G93A mice decreases neuroinflammation, reduces motor neuron death, and increases mice life span, here we examined whether this effect could be mediated by an enhancement of the heat shock response. (2) Methods: Heat shock protein expression was analyzed in vitro and in vivo. Histamine was provided to primary microglia and NSC-34 motor neurons expressing the SOD1-G93A mutation. The brain permeable histamine precursor histidine was chronically administered to symptomatic SOD1-G93A mice. Spine density was measured by Golgi-staining in motor cortex of histidine-treated SOD1-G93A mice. (3) Results: We demonstrate that histamine activates the heat shock response in cultured SOD1-G93A microglia and motor neurons. In SOD1-G93A mice, histidine augments the protein content of GRP78 and Hsp70 in spinal cord and cortex, where the treatment also rescues type I motor neuron dendritic spine loss. (4) Conclusion: Besides the established histaminergic neuroprotective and anti-inflammatory effects, the induction of the heat shock response in the SOD1-G93A model by histamine confirms the importance of this pathway in the search for successful therapeutic solutions to treat ALS.

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Vitronectin is expressed in the ventral region of the neural tube and promotes the differentiation of motor neurons

Development ◽

10.1242/dev.124.24.5139 ◽

1997 ◽

Vol 124 (24) ◽

pp. 5139-5147 ◽

Cited By ~ 7

Author(s):

J.R. Martinez-Morales ◽

J.A. Barbas ◽

E. Marti ◽

P. Bovolenta ◽

D. Edgar ◽

...

Keyword(s):

Motor Neuron ◽

Sonic Hedgehog ◽

Motor Neurons ◽

Neural Tube ◽

Matrix Protein ◽

Floor Plate ◽

Extracellular Matrix Protein ◽

Neuron Differentiation

The extracellular matrix protein vitronectin and its mRNA are present in the embryonic chick notochord, floor plate and in the ventral neural tube at the time position of motor neuron generation. When added to cultures of neural tube explants of developmental stage 9, vitronectin promotes the generation of motor neurons in the absence of either notochord or exogenously added Sonic hedgehog. Conversely, the neutralisation of endogenous vitronectin with antibodies inhibits over 90% motor neuron differentiation in co-cultured neural tube/notochord explants, neural tube explants cultured in the presence of Sonic hedgehog, and in committed (stage 13) neural tube explants. Furthermore, treatment of embryos with anti-vitronectin antibodies results in a substantial and specific reduction in the number of motor neurons generated in vivo. These results demonstrate that vitronectin stimulates the differentiation of motor neurons in vitro and in vivo. Since the treatment of stage 9 neural tube explants with Sonic hedgehog resulted in induction of vitronectin mRNA expression before the expression of floor plate markers, we conclude that vitronectin may act either as a downstream effector in the signalling cascade induced by Sonic hedgehog, or as a synergistic factor that increases Shh-induced motor neuron differentiation.

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Isolation of Mature Spinal Motor Neurons and Single-cell Analysis Using the Comet Assay of Early Low-level DNA Damage Induced In Vitro and In Vivo

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540104900804 ◽

2001 ◽

Vol 49 (8) ◽

pp. 957-972 ◽

Cited By ~ 12

Author(s):

Zhiping Liu ◽

Lee J. Martin

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Motor Neuron ◽

Single Cell ◽

Motor Neurons ◽

Ventral Horn ◽

Motor Neuron Degeneration ◽

Spinal Motor Neurons

We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in α-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration. (J Histochem Cytochem 49:957–972, 2001)

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