Bisperoxovanadium Mediates Neuronal Protection through Inhibition of PTEN and Activation of PI3K/AKT-mTOR Signaling after Traumatic Spinal Injuries

Abstract

Although mechanisms involved in progression of cell death in spinal cord injury (SCI) have been studied extensively, few are clear targets for translation to clinical application. One of the best-understood mechanisms of cell survival in SCI is phosphatidylinositol-3-kinase (PI3K)/Akt and associated downstream signaling. Clear therapeutic efficacy of a phosphatase and tensin homologue (PTEN) inhibitor called bisperoxovanadium (bpV) has been shown in SCI, traumatic brain injury, stroke, and other neurological disease models in both neuroprotection and functional recovery. The present study aimed to elucidate mechanistic influences of bpV activity in neuronal survival in in vitro and in vivo models of SCI. Treatment with 100 nM bpV(pic) reduced cell death in a primary spinal neuron injury model (p < 0.05) in vitro, and upregulated both Akt and ribosomal protein S6 (pS6) activity (p < 0.05) compared with non-treated injured neurons. Pre-treatment of spinal neurons with a PI3K inhibitor, LY294002 or mammalian target of rapamycin (mTOR) inhibitor, rapamycin blocked bpV activation of Akt and ribosomal protein S6 activity, respectively. Treatment with bpV increased extracellular signal-related kinase (Erk) activity after scratch injury in vitro, and rapamycin reduced influence by bpV on Erk phosphorylation. After a cervical hemicontusive SCI, Akt phosphorylation decreased in total tissue via Western blot analysis (p < 0.01) as well as in penumbral ventral horn motor neurons throughout the first week post-injury (p < 0.05). Conversely, PTEN activity appeared to increase over this period. As observed in vitro, bpV also increased Erk activity post-SCI (p < 0.05). Our results suggest that PI3K/Akt signaling is the likely primary mechanism of bpV action in mediating neuroprotection in injured spinal neurons.

Keywords: PTEN; bisperoxovanadium; bpV; mTOR spinal cord injury; neuroprotection.

FIG. 1.

In vitro and in vivo experimental design and timeline. PI, propidium iodide; C5 Hemi-SCI, cervical level 5 hemicontusion spinal cord injury; bpV(pic), dipotassium bisperoxo (picolinato) vanadium.

FIG. 2.

BpV protected spinal neurons from injury in an in vitro model of trauma. (A) Scratch injury produced a graded increase of cell death as indicated by lactase dehydrogenase (LDH) assay (p < 0.01 compared with control). (B) Administration of 100 nM bisperoxovanadium (bpV) significantly reduced LDH release at day one post-scratch injury. (C) This same dose of bpV reduced propidium iodide (PI, red) and Hoechst (blue) nuclear co-labeling at 24 h in spinal neurons after scratch injury, indicating reduced cell death after the bpV treatment. Data were expressed as mean ± standard error of the mean; n = 3 experiments; All data analyzed via one-way analysis of variance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 3.

Bisperoxovanadium (bpV) activated prosurvival kinase/mammalian target of rapamycin (Akt/mTOR) in injured spinal neurons in vitro. (A) Scratch injury induced a decrease in Akt phosphorylation that was significantly reversed by bpV treatment. Application of a PI3K inhibitor LY294002 diminished Akt phosphorylation in the presence of bpV. (B) Ribosomal protein S6 phosphorylation, a marker of mTOR activity, significantly increased after scratch injury as observed in vivo. Treatment with bpV significantly increased p-S6 expression over scratch-induced injury alone. Treatment with an mTOR inhibitor, rapamycin, reduced S6 phosphorylation post-scratch injury and bpV treatment. Data were expressed as mean ± standard error of the mean; n = 3–4 experiments; All data analyzed via one-way analysis of variance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 4.

Bisperoxovanadium (bpV) promoted extracellular signal-related kinase (Erk) activity in an mammalian target of rapamycin (mTOR)-dependent manner. (A) MEK and Erk activity inhibitor, U0126, did not significantly reduce neuron scratch injury-mediated cell death, while bpV did even when combined with U0126. (B) Rapamycin reduced Erk activity, suggesting that the injury induced Erk activation occurred via an mTOR-dependent manner. Data were expressed as mean ± standard error of the mean; n = 3 experiments. All data analyzed via one-way analysis of variance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 5.

Inverse relationship between phosphatase and tensin homologue (PTEN) activity and prosurvival kinase (p-Akt) expression after a cervical hemi-contusion spinal cord injury (SCI). (A) The PTEN phosphorylation was considerably reduced by day three post-SCI, indicating PTEN phosphatase activity increased over this period. (B) Conversely, Akt phosphorylation significantly decreased starting at day one post-SCI. (C) Representative Western blots of PTEN, p-PTEN, p-Akt, and Akt. β-tubulin served as a loading control. Data were expressed as mean ± standard error of the mean; n = 4–5/time point; All data analyzed via one-way analysis of variance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 6.

Ventral horn motor neuron prosurvival kinase (p-Akt) expression mirrored total spinal tissue p-Akt expression post-spinal cord injury (SCI). (A) Illustration of region of neuronal imaging and quantification. (B) As shown for total spinal tissue, Akt phosphorylation decreased in ventral horn neurons throughout the first week post-SCI. (C) Ventral horn neurons were significantly decreased in the penumbral region beginning one day post-injury, but sustained this level throughout the first week after SCI. (D) Representative double immunofluorescence labeling shows decreased Akt phosphorylation in the ventral horn motor neurons over time post-injury. Data were expressed as mean ± standard error of the mean; n = 3/ time point; All data analyzed via one-way analysis of variance; *p < 0.05 compared with sham.

FIG. 7.

Extracellular signal-related kinase (Erk) and p-S6 colocalized and showed similar expression profile in the spinal cord after injury. (A–F) Both p-Erk and p-S6 were highly expressed in the ventral horn motor neurons at 24 h post-SCI compared with the sham control (white arrows). (G,I) The Erk activity and (H,I) p-S6 showed similarly expressional changes over time in the total spinal cord tissue. (I) Representative Western blot images for p-S6, p-Erk, and Erk. β-tubulin served as a loading control. Data were expressed as mean ± standard error of the mean; n = 3 for immunofluorescence double labeling. n = 4–5/time point for Western blot. All data analyzed via one-way analysis of variance; n = 3 for immunofluorescence double labeling. n = 4–5/ time point for Western blot. *p < 0.05; **p < 0.01; ***p < 0.001 compared with sham.

FIG. 8.

Bisperoxovanadium (bpV) reduced apoptosis, and enhanced extracellular signal-related kinase (Erk) and glycogen synthase kinase (GSK) 3β activation after spinal cord injury (SCI). (A) Treatment with bpV significantly decreased SCI-induced elevation in caspase 3 activity at one day post-injury. Simultaneously, (B) GSK3β phosphorylation, a marker of Akt activity, and (C) Erk activity were increased by bpV in injured spinal tissue. (D) Representative Western blots of p-GSK3β, cleaved caspase 3, p-Erk, and Erk. β-tubulin served as a loading control. Data were expressed as mean ± standard error of the mean; n = 4–6 for Western blot. All data analyzed via one-way analysis of variance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 9.

Schematic diagram for proposed mechanisms of injury and bisperoxovanadium (bpV)-induced signaling changes in vivo and in vitro. Based on our findings, we believe bpV-mediated neuroprotection in spinal cord injury and an in vitro model of spinal neuron injury primarily through PI3K/Akt/mTOR signaling. The activation of extracellular signal-related kinase (Erk) also appears to play a role, although this appears to be mediated through cross-talk with mTOR. Akt/mTOR, prosurvival kinase/mammalian target of rapamycin; PTEN, phosphatase and tensin homologue.