Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo

AbstractDegeneration and death of motor neurons in Amyotrophic Lateral Sclerosis (ALS) are associated with increased lipid peroxidation. Lipid peroxidation is the driver of ferroptosis, an iron-dependent oxidative mode of cell death. However, the importance of ferroptosis in motor neuron degeneration of ALS remains unclear. Glutathione peroxidase 4 (Gpx4) is a key enzyme in suppressing ferroptosis by reducing phospholipid hydroperoxides in membranes. To assess the effect of increased protection against ferroptosis on motor neuron disease, we generated SOD1G93AGPX4 double transgenic mice by cross-breeding GPX4 transgenic mice with SOD1G93A mice, a widely used ALS mouse model. Compared with control SOD1G93A mice, both male and female SOD1G93AGPX4 mice had extended lifespans. SOD1G93AGPX4 mice also showed delayed disease onset and increased motor function, which were correlated with ameliorated spinal motor neuron degeneration and reduced lipid peroxidation. Moreover, cell toxicity induced by SOD1G93A was ameliorated by Gpx4 overexpression and by chemical inhibitors of ferroptosis in vitro. We further found that the anti-ferroptosis defense system in spinal cord tissues of symptomatic SOD1G93A mice and sporadic ALS patients might be compromised due to deficiency of Gpx4. Thus, our results suggest that ferroptosis plays a key role in motor neuron degeneration of ALS.

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PAK4 Suppresses Motor Neuron Degeneration in hSOD1G93A -Linked ALS Cell and Rat Models

10.21203/rs.3.rs-93482/v1 ◽

2020 ◽

Author(s):

Chaohua Cong ◽

Weiwei Liang ◽

Chunting Zhang ◽

Ying Wang ◽

Yueqing Yang ◽

...

Keyword(s):

Motor Neuron ◽

Western Blotting ◽

Motor Neurons ◽

Neuroprotective Effect ◽

Tunel Staining ◽

Motor Neuron Degeneration ◽

Neuron Degeneration ◽

In Vivo Models ◽

Creb Pathway

Abstract Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. The exact mechanisms underlying motor neuron death in ALS are still not fully understood, but a growing body of evidence indicates that inflammatory could accentuate disease severity and accelerate disease progression. Currently, no neuroprotective strategies have effectively prevented the progression of this disease.Methods: IF, western blotting and RT-PCR were used to analyze the expression of PAK4 in vitro and in vivo models of ALS. We examined PAK4 function in ALS and the underlying mechanism by cell transfection, intraspinally injection of LV-PAK4 in hSOD1G93A mice, flow cytometry, TUNEL staining, IF and western blotting.Results: Here, we observed that the expression and activity of PAK4 significantly decreased in hSOD1G93A-related cell and mouse models of ALS. In hSOD1G93A mice，the expression of PAK4 began to decrease at early-symptom stages of the disease. PAK4 silencing increased degeneration of motor neurons (NSC34 cells) and suppressed the CREB pathway. Overexpression of PAK4 protected motor neurons from hSOD1G93A-induced degeneration by increasing the levels and transcriptional activity of CREB. The neuroprotective effect of PAK4 was markedly inhibited by compound 3i, a specific CREB inhibitor. In hSOD1G93A-linked cell and mice, the CREB pathway, as the downstream target of decreased PAK4, was inhibited, and cell apoptosis increased. We also found that the expression of PAK4 was negatively regulated by miR-9-5p, and the miR-9-5p levels were upregulated in ALS. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1G93A mice suppressed motor neuron degeneration, prolonged survival and promoted the CREB pathway. Conclusion: These results indicate that PAK4 plays a protective role for motor neurons by targeting CREB, suggesting it may be a useful therapeutic target in ALS.

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In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice

Human Molecular Genetics ◽

10.1093/hmg/11.14.1615 ◽

2002 ◽

Vol 11 (14) ◽

pp. 1615-1625 ◽

Cited By ~ 44

Author(s):

J.-C. Lesbordes

Keyword(s):

Skeletal Muscle ◽

Motor Neuron ◽

Motor Neuron Degeneration ◽

Neuron Degeneration

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Astrocytes carrying the superoxide dismutase 1 (SOD1G93A) mutation induce wild-type motor neuron degeneration in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1103141108 ◽

2011 ◽

Vol 108 (43) ◽

pp. 17803-17808 ◽

Cited By ~ 109

Author(s):

S. T. Papadeas ◽

S. E. Kraig ◽

C. O'Banion ◽

A. C. Lepore ◽

N. J. Maragakis

Keyword(s):

Superoxide Dismutase ◽

Motor Neuron ◽

Superoxide Dismutase 1 ◽

Wild Type ◽

Motor Neuron Degeneration ◽

Neuron Degeneration ◽

Type Motor

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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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A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin

Scientific Reports ◽

10.1038/s41598-021-97405-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Phaneendra Chennampally ◽

Ambreen Sayed-Zahid ◽

Prabakaran Soundararajan ◽

Jocelyn Sharp ◽

Gregory A. Cox ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Mouse Model ◽

Motor Neuron ◽

Motor Neurons ◽

Embryonic Stem ◽

Immunosuppressive Drug ◽

Motor Neuron Degeneration ◽

Neuron Degeneration ◽

Lateral Sclerosis

AbstractTAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

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