Purification and Culture of Spinal Motor Neurons

Graber, David J.; Harris, Brent T.

doi:10.1101/pdb.top070920

Cited by 2 publications

(1 citation statement)

References 11 publications

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“…Embryonic motor neuron pellets were gently resuspended at 200,000 cells/cm 2 in freshly prepared Motor Neuron Growth Medium (MNGM), which is described in detail by Graber DJ et al (Graber and Harris, 2013). Briefly, the MNGM consists of Neurobasal A medium (NB-medium, Life Technologies) supplemented with 1X B-27 Serum-Free Supplement (Gibco/Life Technologies), 1X SATO supplement, 5 μg mL −1 Insulin (Gibco/Life Technologies), 1 mM Sodium pyruvate (Gibco/Life Technologies), 2 mM L-Glutamine (Gibco/Life Technologies), 40 ng mL −1 of 3,3,5-triiodo-L-thyronine sodium salt (T3; Sigma-Aldrich), 1 μg mL −1 Mouse laminin (Gibco/Life Technologies), 417 ng mL −1 Forskolin (Sigma-Aldrich), 5 μg mL −1 N-acetyl-L-cysteine (NAC, Sigma-Aldrich) and 1x Penicillin-streptomycin (Gibco/Life Technologies).…”

Section: Cell Culturementioning

confidence: 99%

ALS-linked SOD1 Mutants Enhance Outgrowth, Branching, and the Formation of Actin-Based Structures in Adult Motor Neurons

Osking

Ayers

Hildebrandt

et al. 2018

Preprint

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Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron cell death and subsequent paralysis of voluntary muscles. Although ALS specifically affects motor neurons, some cells are resistant to disease progression. Most ALS studies have focused on the cellular mechanisms that cause loss of motor neuron viability. Less is known about the surviving neurons, and most of that information has come from gene expression profiling. In this study, we functionally characterize the surviving spinal motor neurons by culturing them from SOD1 ALS mouse models at various stages of disease progression. Surprisingly, we found that in comparison to non-transgenic controls, ALS resistant motor neurons from adult SOD1 G93A mice have enhanced axonal outgrowth and dendritic branching. Further, the enhanced outgrowth occurs before the mice become symptomatic, but increases with disease progression. Motor neurons from SOD1 G93A mice also display an increase in the number and size of actin-based structures such as growth cones and filopodia. The increased outgrowth and branching phenotype is predominantly cellintrinsic and can be induced in motor neurons from non-transgenic mice by exogenous expression of SOD1 G93A . These results indicate that expression of mutant SOD1 in ALS-resistant adult motor neurons can enhance their regenerative capability via a mechanism that is not directly correlated with the onset of ALS symptoms. Understanding the positive effects that mutant SOD1 has on motor neuron regeneration could lead to new therapeutic strategies that capitalize on this mechanism.

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Section: Cell Culturementioning

confidence: 99%

ALS-linked SOD1 Mutants Enhance Outgrowth, Branching, and the Formation of Actin-Based Structures in Adult Motor Neurons

Osking

Ayers

Hildebrandt

et al. 2018

Preprint

View full text Add to dashboard Cite

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ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway

et al. 2017

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Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93A transgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+ concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS.

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Purification and Culture of Spinal Motor Neurons

Cited by 2 publications

References 11 publications

ALS-linked SOD1 Mutants Enhance Outgrowth, Branching, and the Formation of Actin-Based Structures in Adult Motor Neurons

ALS-linked SOD1 Mutants Enhance Outgrowth, Branching, and the Formation of Actin-Based Structures in Adult Motor Neurons

ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway

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