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

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.

Download Full-text

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

10.1101/835082 ◽

2019 ◽

Cited By ~ 4

Author(s):

Laura Fumagalli ◽

Florence L. Young ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Arpan R. Mehta ◽

...

Keyword(s):

Axonal Transport ◽

Motor Neurons ◽

Single Molecule ◽

Induced Pluripotent Stem Cell ◽

Cytoplasmic Dynein ◽

Inhibitory Interaction ◽

Hexanucleotide Repeat ◽

Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

Download Full-text

Real time imaging reveals a peroxisomal reticulum in living cells

Journal of Cell Science ◽

10.1242/jcs.113.20.3663 ◽

2000 ◽

Vol 113 (20) ◽

pp. 3663-3671 ◽

Cited By ~ 1

Author(s):

M. Schrader ◽

S.J. King ◽

T.A. Stroh ◽

T.A. Schroer

Keyword(s):

Real Time ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Real Time Imaging ◽

Peroxisomal Targeting ◽

Microtubule Motor ◽

Targeting Signal ◽

Peroxisomal Targeting Signal

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

Download Full-text

“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.

Download Full-text

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.

Download Full-text

PACAP Modulates the Autophagy Process in an In Vitro Model of Amyotrophic Lateral Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms21082943 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2943 ◽

Cited By ~ 4

Author(s):

Agata Grazia D’Amico ◽

Grazia Maugeri ◽

Salvatore Saccone ◽

Concetta Federico ◽

Sebastiano Cavallaro ◽

...

Keyword(s):

Cell Death ◽

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

In Vitro Model ◽

Erk Signaling ◽

Hypoxic Stress ◽

Vitro Model ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology leading to motor neuron degeneration. Many gene alterations cause this pathology, including mutation in Cu, Zn superoxide dismutase (SOD1), which leads to its gain of function. Mutant SOD1 proteins are prone to aberrant misfolding and create aggregates that impair autophagy. The hypoxic stress is strictly linked to the disease progression since it induces uncontrolled autophagy activation and the consequent high rates of cell death. Previously, we showed that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activity in cultured mSOD1 motor neurons exposed to serum deprivation. To date, no studies have examined whether the protective effect of PACAP on mSOD1 cells exposed to hypoxic insult is mediated through the regulation of the autophagy process. In the present study, we used the neuroblastoma-spinal cord-34 (NSC-34) cell line, stably expressing human wild type or mutant SOD1 G93A, to represent a well characterized in vitro model of a familial form of ALS. These cells were exposed to 100-µM desferrioxamine mesylate salt for 24h, to mimic the hypoxic stress affecting motor neurons during the disease progression. Our results showed that PACAP treatment significantly reduced cell death and hypoxia-induced mSOD1 accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators. These results were also confirmed by evaluating the vacuole formation detected through light chain 3 (LC3) immunofluorescence. Furthermore, the PACAP effects on autophagy seem to be mediated through the activation of the MAPK/ERK signaling pathway. Overall, our data demonstrated that PACAP exerts an ameliorative effect on the mSOD1 motor neuron viability by modulating a hypoxia-induced autophagy process through activation of MAPK/ERK signaling cascade.

Download Full-text

The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

10.1101/232652 ◽

2017 ◽

Author(s):

Kuo-Fu Tseng ◽

Pan Wang ◽

Yuh-Ru Julie Lee ◽

Joel Bowen ◽

Allison M. Gicking ◽

...

Keyword(s):

Coiled Coil ◽

Preprophase Band ◽

Cytoplasmic Dynein ◽

Coiled Coils ◽

Land Plants ◽

Actin Binding ◽

Actin Binding Domain ◽

Microtubule Motor

AbstractIn animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

Download Full-text

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

10.1101/2021.11.23.469650 ◽

2021 ◽

Author(s):

Brigid K Jensen ◽

Kevin J McAvoy ◽

Nicolette M Heinsinger ◽

Angelo C Lepore ◽

Hristelina Ilieva ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Neutralizing Antibodies ◽

Rna Binding ◽

Neuronal Damage ◽

Specific Expression ◽

Fused In Sarcoma ◽

Knockout Animals

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.

Download Full-text

Fructose-a double edged sword for cell death versus differentiation and long-term survival of NSC-34 motor neuron like cells in vitro

10.21203/rs.3.rs-181783/v1 ◽

2021 ◽

Author(s):

Divya Lodha ◽

Jamuna R. Subramaniam

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Motor Neurons ◽

Response Regulator ◽

Nuclear Staining ◽

Term Survival ◽

High Fructose

Abstract In various neurological and neurodegenerative diseases (ND), motor neurons (MN) of the spinal cord are affected leading to movement impairments. The ND, Amyotrophic Lateral Sclerosis (ALS), is caused due to MN degeneration. ALS afflicts athletes and other major sports personalities, who generally consume fructose enriched sports drinks. Recently, we have reported that high fructose (F5%) impairs the metabolic activity in the NSC-34, MN cell line and reduces the healthspan of C. elegans. But how fructose impacts the MNs either in vitro or in vivo in the long term is not understood. Here we report, to our surprise, that high fructose (F5%) treatment of NSC-34 leads to differentiation of 1-2% of cells with progressive neurite extension. They could be maintained for 80 days in vitro with 5% CO2 and O2 at 18.8%. On the contrary, 5% fructose significantly reduced cell viability by ~85% and inhibited cell proliferation by Day10. Nuclear staining displayed multiple nuclei in the cells indicative of cytokinesis inhibition which led to the lack of cell proliferation. Further, F5% significantly increased ROS levels (^~34%), the potential cause for reduced viability. In addition, no induction of expression of the master oxidative stress response regulator, the transcription factor, nrf-2, or the downstream effector, sod1, was evident. Despite the adverse effects, in the absence of any, F5% is a potential strategy to maintain at least a small percentage of MNs for a long time, ~45 days in vitro, which also reinforces the Redox-Cell death versus cell survival conundrum.

Download Full-text

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.

Download Full-text

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.

Download Full-text