Differentiation of Human Neural Stem Cells into Motor Neurons Stimulates Mitochondrial Biogenesis and Decreases Glycolytic Flux

Abstract

Differentiation of human pluripotent stem cells (hPSCs) in vitro offers a way to study cell types that are not accessible in living patients. Previous research suggests that hPSCs generate ATP through anaerobic glycolysis, in contrast to mitochondrial oxidative phosphorylation (OXPHOS) in somatic cells; however, specialized cell types have not been assessed. To test if mitobiogenesis is increased during motor neuron differentiation, we differentiated human embryonic stem cell (hESC)-and induced pluripotent stem cell-derived human neural stem cells (hNSCs) into motor neurons. After 21 days of motor neuron differentiation, cells increased mRNA and protein levels of genes expressed by postmitotic spinal motor neurons. Electrophysiological analysis revealed voltage-gated currents characteristic of excitable cells and action potential formation. Quantitative PCR revealed an increase in peroxisome proliferator-activated receptor gamma coactivator 1-a (PGC-1a), an upstream regulator of transcription factors involved in mitobiogenesis, and several of its downstream targets in hESC-derived cultures. This correlated with an increase in protein expression of respiratory subunits, but no increase in protein reflecting mitochondrial mass in either cell type. Respiration analysis revealed a decrease in glycolytic flux in both cell types on day 21 (D21), suggesting a switch from glycolysis to OXPHOS. Collectively, our findings suggest that mitochondrial biogenesis, but not mitochondrial mass, is increased during differentiation of hNSCs into motor neurons. These findings help us to understand human motor neuron mitobiogenesis, a process impaired in amyotrophic lateral sclerosis, a neurodegenerative disease characterized by death of motor neurons in the brain and spinal cord.

The paradox of metabolism in quiescent stem cells

“…Constitutive expression of HK2 and LDHA during differentiation resulted in cell death specifically in the differentiated neurons, which demonstrates that downregulation of aerobic glycolysis is required for proper differentiation . Similarly, another study found that differentiation of human neural stem cells into postmitotic motor neurons is associated with decreased glycolytic flux and increased mitochondrial biogenesis . As described below, however, another study of neural stem cell metabolism came to a different conclusion and determined that neural stem cells rely on fatty acid oxidation rather than glycolysis to survive .…”

Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development

“…Lange and colleagues have shown that increased oxygen tension in the NSC niche inactivates HIF1α, resulting in differentiation to neuronal and glial fates [26]. Not surprisingly, embryonic neural progenitor cells (NPCs) utilize aerobic glycolysis and switch to OxPhos during neuronal differentiation [27,28]. This correlates with up-regulation of PGC1α which induces mitochondrial biogenesis and the establishment of OxPhos [28].…”

“…Not surprisingly, embryonic neural progenitor cells (NPCs) utilize aerobic glycolysis and switch to OxPhos during neuronal differentiation [27,28]. This correlates with up-regulation of PGC1α which induces mitochondrial biogenesis and the establishment of OxPhos [28]. As adult neural stem cells differentiate in vitro to neurons they also decrease aerobic glycolysis though a HIF1α-independent mechanism [25].…”

Energy metabolism in neuronal/glial induction and in iPSC models of brain disorders

“…During differentiation of human NPCs into motor neurons, mitochondrial biogenesis is elevated while mitochondrial mass remains unchanged [78]. Differentiating cells increase their mitochondrial components, leading to enhanced bioenergetic capacity.…”

“…Differentiating cells increase their mitochondrial components, leading to enhanced bioenergetic capacity. Hence, the generation of motor neurons from NPCs requires ATP synthesis coupling and low glycolytic flux [78].…”