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Drugging Resident Cellular Progenitors for Spinal Cord Injury Repair

Original article from STEM CELLS

FM19G11 Favors Spinal Cord Injury Regeneration and Stem Cell Self-Renewal by Mitochondrial Uncoupling and Glucose Metabolism Induction

Activation of resident adult stem/progenitor cells for the treatment of some disease/injury states is often seen as a viable alternative to the transplantation of embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) derived cells. Ependymal stem progenitor cells (epSPCs) reside in the adult spinal cord and, upon transplantation, have been shown to rescue lost locomotor activity associated with spinal cord injury (SCI) (Moreno-Manzano et al 2009). Therefore, activation of resident epiSCs through pharmacological means may be a viable non-invasive therapeutic option for SCI.   Previous research has provided potential targets for therapeutic agents; mitochondrial bio-energetics have been identified as a potentially drugable target for SCI (Sullivan et al), with mitochondrial uncoupling a suggested  pharmacological therapy for acute SCI (Patel et al) or those patients with long term injury (Jin et al). Additionally, glucose uptake and ATP generated by glycolysis is abundant in stem cells, but not in differentiated cells, suggesting that modulation of glucose uptake and trafficking may also represent a target to activate adult stem cells. Now in a study published in Stem Cells, researchers from the laboratory of Victoria Moreno-Manzano at theCIPF, Valencia, Spain have shown that the chemical compound FM19G11, first identified as an inhibitor of HIFα protein expression and transcriptional activity under hypoxic conditions and which represses stemness-associated genes (Moreno-Manzano et al 2010), can modify the mitochondrial uncoupling process and induces glucose uptake by activation of AMPK (AMP-activated kinase) and AKT (or protein kinase B) signaling pathways in epSPCs. Excitingly, this is associated with an increase in the self-renewal of epSPC and the rescue of lost locomotor activity in rat SCI indicating this drugs potential usefulness for spinal cord disorders (Rodríguez-Jimnez et al).

Initial experiments compared the gene expression profile of rat epSPCs treated with FM19G11 with control DMSO treated epSPCs. This demonstrated that genes over-expressed in the FM19G11-treated cells were associated with ATP-biosynthetic processes, including the induction of the mitochondrial uncoupling proteins UCP1 and UCP2. Further cellular analysis found that ATP consumption rose quickly following FM19G11 treatment and then fell over a period of 1-2 hours, accompanied by a reduction of mitochondrial activity up to 4 hours. However, at 24-48 hours post treatment, cellular ATP levels rose alongside an increase in mitochondrial activity and an increase in mitochondrial biosynthesis. Rapid phosphorylation of AMPK and AKT was also caused by FM19G11 treatment leading to the induction of downstream targets such as cyclic-AMP and p-CREB (cAMP response element-binding protein). In addition, protein expression of the glucose transporter gene GLUT4 and its location to the cytosolic membrane was induced, with PI3K/AKT- and AMPK-mediated signaling known to act as a regulatory mechanism for the activity of glucose transporters (Okada et al). These pathways were shown to be involved in regulating glucose uptake through the use of specific inhibitors, Wortmannin (PI3K/AKT inhibitor) or Compound C (AMPK inhibitor), both of which inhibited glucose uptake in the presence of FM19G11. Additionally, mitochondrial biosynthesis was blocked by the reduction of GLUT4 by RNAi in epSPCs.

Subsequent neurosphere assays found a significant increase in epSPC proliferation upon FM19G11 treatment, which was confirmed through the immunodetection of higher levels of the mitotic marker phospho-histone H3. TERT expression and telomerase activity were also induced in epSPCs treated with FM19G11, as were Sox2, Oct4 and Notch1 expression. Loss of GLUT4 by siRNA however, stopped FM19G11 from inducing Sox2, Oct4 and Notch1 expression. FM19G11-mediated induction of Sox2 and Oct4 was also blocked by the use of inhibitors of AKT (Wortmannin), mTOR (Rapamycin) and AMPK (Compound C), while no induction of Sox2 was observed when UCP2 was ablated by siRNA.

Next, FM19G11 was administered to rats in the lesion area immediately after SCI via an osmotic pump and for the first week after SCI. Excitingly, analysis of locomotor activity found a significant increase in locomotion recovery of hind limbs in animals treated with FM19G11 4 weeks after injury. Analysis of the damaged medullar tissue found no alterations in UCP1 expression, but UCP2, Sox2, Oct4 and Notch1 were all significantly upregulated in the FM19G11 treated mice. Further analysis of the damaged area through in vivo nuclear magnetic resonance (NMR) found that at 1 week, the injury area was smaller with FM19G11 treatment than control, however at 4 weeks when the animals were sacrificed and the lesioned spinal cords analyzed, no significant differences were found either for the quantification of scar area or in cyst/cavity formation. However, TUJ1, a protein that contributes to microtubule stability in neuronal cell bodies and axons with a relevant role in axonal transport, marked more axons crossing the injured area in FM19G11-treated rats than the control, while Vimentin, a neuronal precursor marker that stains the ependymal cell population, was also seen at higher levels in the FM19G11-treated rats.

This report suggests that FM19G11 treatment induced functional recovery after spinal cord injury, through a suggested increment in the self-renewal capacity of ependymal progenitor stem cells by the early induction of a glycolytic-related response associated to a PI3K/AKT signaling induction. Therefore FM19G11 treatment may be a non-invasive, and seemingly non-toxic, method to aid recovery from spinal cord injury through the activation of resident adult stem cells.

 

References

Jin Y et al.
The mitochondrial uncoupling agent 2,4-dinitrophenol improves mitochondrial function, attenuates oxidative damage, and increases white matter sparing in the contused spinal cord.
J Neurotrauma 2004;2110:1396–1404.

Moreno-Manzano V et al.
Activated spinal cord ependymal stem cells rescue neurological function.
Stem Cells 2009;273:733–743.

Moreno-Manzano V et al.
FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects stem cell differentiation status.
J Biol Chem 2010;2852:1333–1342.

Okada T et al.
Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin.
J Biol Chem 1994;2695:3568–3573.

Patel SP et al.
Differential effects of the mitochondrial uncoupling agent, 2,4-dinitrophenol, or the nitroxide antioxidant, Tempol, on synaptic or nonsynaptic mitochondria after spinal cord injury.
J Neurosci Res 2009;871:130–140.

Rodríguez-Jimnez FJ et al.
FM19G11 Favors Spinal Cord Injury Regeneration and Stem Cell Self-Renewal by Mitochondrial Uncoupling and Glucose Metabolism Induction.
Stem Cells. 2012 Oct;30(10):2221-33

Sullivan PG et al.
Temporal characterization of mitochondrial bioenergetics after spinal cord injury.
J Neurotrauma 2007;246:991–999.

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.