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Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons
Published on: January 7, 2019
Extracellular mutant SOD1 induces microglial-mediated motoneuron injury
Weihua Zhao1, David R Beers, Jenny S Henkel
1Department of Neurology, Methodist Neurological Institute, The Methodist Research Institute, The Methodist Hospital, Houston, Texas, USA.
Abstract:
Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase-1 (mSOD1) cause the non-cell-autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1-activated microglia. To dissociate these potential mSOD1-mediated neurotoxic mechanisms, the effects of extracellular human mSOD1(G93A) or mSOD1(G85R) were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1(G93A) did not cause detectable direct killing of motoneurons. In contrast, mSOD1(G93A) or mSOD1(G85R) did induce the morphological and functional activation of microglia, increasing their release of pro-inflammatory cytokines and free radicals. Furthermore, only when microglia was co-cultured with motoneurons did extracellular mSOD1(G93A) injure motoneurons. The microglial activation mediated by mSOD1(G93A) was attenuated using toll-like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1(G93A) is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS.
Insights
Mutant SOD1 (mSOD1) causes motor neuron death in ALS indirectly. Extracellular mSOD1 activates microglia, which then injure motor neurons via TLR pathways, not direct toxicity.
Area of Science:
- Neuroscience
- Immunology
- Genetics
Background:
- Dominant mutations in copper/zinc superoxide dismutase-1 (mSOD1) are linked to inherited amyotrophic lateral sclerosis (ALS).
- Microgliosis, or the activation of microglia, is a key pathological feature observed at sites of motor neuron damage in ALS.
- It remains unclear if extracellular mSOD1 directly harms motor neurons or if it acts indirectly through microglial activation.
Purpose of the Study:
- To investigate the mechanisms by which extracellular mutant SOD1 (mSOD1) causes motor neuron injury in ALS.
- To differentiate between direct neurotoxicity of mSOD1 on motor neurons and mSOD1-mediated microglial activation leading to neuroinflammation.
Main Methods:
- Primary cultures of motor neurons and microglia were utilized to assess the effects of extracellular human mSOD1(G93A) and mSOD1(G85R).
- Experiments involved co-culturing motor neurons and microglia to observe interactions and effects of mSOD1.
- Blocking antibodies for toll-like receptors (TLR) 2, TLR4, and CD14 were used to investigate the role of these pathways in mSOD1-induced microglial activation.
Main Results:
- Exogenous mSOD1(G93A) did not directly kill motor neurons in primary culture.
- Extracellular mSOD1(G93A) and mSOD1(G85R) induced morphological and functional activation of microglia, increasing pro-inflammatory cytokine and free radical release.
- Motor neuron injury occurred only when microglia were co-cultured with motor neurons and exposed to extracellular mSOD1(G93A).
- Microglial activation by mSOD1(G93A) was significantly reduced by blocking TLR2, TLR4, and CD14, or in microglia lacking CD14 expression.
Conclusions:
- Extracellular mSOD1 is not directly toxic to motor neurons in ALS.
- Motor neuron injury by extracellular mSOD1 requires microglial activation.
- The innate immune pathways involving CD14 and toll-like receptors (TLR) mediate mSOD1-induced microglial toxicity, suggesting potential therapeutic targets for ALS.
