Characterization and Modulation of PI3K-Akt Signaling Following Contusive SCI

poster abstractSpinal cord injury (SCI) is devastating, with most injuries being contusive/compressive injuries at the cervical spinal level. There are two mechanisms of damage after acute contusive SCI: a primary mechanical insult to the cord, and secondary injury induced by many biological events, including inflammation and signal-mediated cell death. The extent of tissue damage correlates with functional loss after SCI, therefore it is critical to protect neural tissue for preservation of functional ability. Focusing on cellular signaling events following SCI is a promising direction of investigation, as modulation of such pathways can promote neuroprotection or regeneration following injury. Two particular signaling pathways have been highlighted as mediators of cellular survival post-central nervous system (CNS) injury, the MEK-Erk and PI3K-Akt pathways. Reducing Erk activity has been shown to promote neuroprotection and reduced reactive gliosis, while reduction of PI3K-Akt signaling likely results in initiation of cellular death. Recent studies have demonstrated promotion of regrowth of adult corticospinal (CST) neurons and protection of motor neuron atrophy by disinhibition of PI3K via PTEN deletion or knock-down in these cells. Understanding the signal pathways and mechanisms involved in different cell types, when such response occurs, and the potential interaction between pathways is essential for maximizing development of optimal approaches to treatment following SCI. This study highlights PI3K-Akt signaling involvement following injury, with future directions aimed at better understanding this pathway for targeting therapies to mediate anatomical and functional preservation and recovery following SCI

Systemic Bisperoxovanadium Activates Akt/mTOR, Reduces Autophagy, and Enhances Recovery following Cervical Spinal Cord Injury

<div><p>Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post-spinal cord injury, and sheds light on the signaling involved in its action.</p> </div

Effects of bpV(pic) on mTOR and autophagy-related protein analysis 1d post-SCI.

<p>A) Western blot profiles from tissue collected from experimental animals 1d post-SCI. B–E) Quantification of blots shown in A. B) Total PTEN protein expression does not significantly change following injury, though a mild increase in expression is observed. C) p-Akt levels significantly decrease following injury, and are significantly increased following bpV (pic) treatment. D) Downstream, p-S6 protein levels significantly increase following injury, and are enhanced further following bpV treatment. E) LC3 II ratio to LC3 I, an indicator of autophagic activity, is increased following injury, and is significantly reduced following bpV(pic) therapy. **, <i>p</i><0.01; *, <i>p</i><0.05. <i>n</i> = 4−5 per group. Error bars = SEM.</p

bpV-treatment enhances forelimb functional recovery.

<p>A) By 6 weeks post-injury, bpV(pic)-treated rats exhibited significantly enhanced forelimb function over vehicle-treated animals as scored using an 8-point treat manipulation behavioral scale (6.8 vs. 4.5). Sham animals scored at or near perfect (8 points) throughout the study. B) Testing scores showed a positive linear correlation with arm joint articulation ability (<i>R²</i> = 0.88). C) Images portraying a rat grasping and manipulating a flavored cereal ring, the treat used in this assessment. **, <i>p</i><0.01; *, <i>p</i><0.05. <i>n</i> = 4−5. Error bars = SEM.</p

bpV(pic) reduces neuronal autophagosome aggregation.

<p>Vehicle-treated rats exhibited dense intracellular LC3-positive autophagosome aggregation in neurons (arrows). Treatment with bpV promoted a decrease in neuronal autophagosome clustering. <i>n</i> = 3. Scale bar = 50 µm.</p

Phospho-S6 cellular localization following injury.

<p>Like PTEN, phospho-S6 (p-S6) colocalized with many cell and structure types 1d following SCI. p-S6 was expressed very abundantly in A–F) neurons, and G–L) hypertrophic astrocytes following SCI, as well as M–P) oligodendrocytes, as indicated by arrows. <i>n</i> = 3. Scale bar = 100 µm.</p

Potential mechanistic explanation for bpV(pic)-mediated neuroprotective effects.

<p>PTEN's phosphatase activity converts phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) into phosphatidylinositol (4,5)-bisphosphate (PIP₂), thus inhibiting downstream Akt and mTOR signaling. PI3K converts PIP₂ into PIP₃, which can then activate Akt and mTOR, thus enhancing p-S6 expression and contributing to the decrease in cellular autophagic activity that may be involved in programmed cell death, and leading to neuroprotection.</p

Acute bpV therapy reduces motor neuron loss following SCI.

<p>A) bpV(pic) treatment spared, on average, more ventral horn motor neurons than vehicle-treatment. Significantly more motor neurons were present in the ventral horn 2 mm rostral and caudal to the lesion epicenter of bpV(pic) treated animals. B & C) Photomicrographical representation showing Nissl-eosin stained ventral horns of spinal tissue extracted 6 weeks post-SCI. Sections shown are from 2 mm rostral to the epicenter and demonstrate the increase in motor neurons in bpV-treated animals over vehicle-treatment. *, <i>p</i><0.05. <i>n</i> = 4−5. Error bars = SEM. Scale bar = 150 µm.</p

bpV(pic) reduces lesion size and cavitation following C5 hemicontusion SCI.

<p>A) Tissue extracted from rats 6 wks post-SCI demonstrate reduced lesion and cavitation through Nissl-eosin staining compared to vehicle treated animals. B & C) Graphical representation showing statistically significant reduction in lesion and cavity volumes, and increased spared tissue following bpV(pic) treatment. D) 3D-reconstruction from representative cases illustrating the neuroprotective effects of acute bpV(pic) therapy. **, <i>p</i><0.01; *, <i>p</i><0.05. <i>n</i> = 4−5. Error bars = SEM. Scale bar = 1 mm.</p