Abstract
During the progression of the neurodegenerative process, mitochondria participates in several intercellular signaling pathways. Voltage-dependent anion-selective channel 1 (VDAC1) is a mitochondrial porin involved in the cellular metabolism and apoptosis intrinsic pathway in many neuropathological processes. In spinal cord injury (SCI), after the primary cell death, a secondary response that comprises the release of pro-inflammatory molecules triggers apoptosis, inflammation, and demyelination, often leading to the loss of motor functions. Here, we investigated the functional role of VDAC1 in the neurodegeneration triggered by SCI. We first determined that in vitro targeted ablation of VDAC1 by specific morpholino antisense nucleotides (MOs) clearly promotes neurite retraction, whereas a pharmacological blocker of VDAC1 oligomerization (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid, DIDS), does not cause this effect. We next determined that, after SCI, VDAC1 undergoes conformational changes, including oligomerization and N-terminal exposition, which are important steps in the triggering of apoptotic signaling. Considering this, we investigated the effects of DIDS in vivo application after SCI. Interestingly, blockade of VDAC1 oligomerization decreases the number of apoptotic cells without interfering in the neuroinflammatory response. DIDS attenuates the massive oligodendrocyte cell death, subserving undisputable motor function recovery. Taken together, our results suggest that the prevention of VDAC1 oligomerization might be beneficial for the clinical treatment of SCI.
Highlights
Spinal cord injury (SCI) can affect mobility and sensibility, first mainly due to axonal rupture and secondly, due to tissue environment toxicity and the consequent inflammatory response
Voltage-dependent anion-selective channel 1 (VDAC1) allows the passage of several metabolites, such as pyruvate, succinate, malate, reduced nicotinamide adenine dinucleotide (NADH), reactive oxygen species (ROS)[21,26,27], and ATP28, in addition to specific ions, including Na+, K+, Ca2+ and Cl−26,29,30. 3D-structures of recombinant VDAC1 obtained by X-ray crystallography revealed that its structure is composed of 19 β-strands arranged as a barrel[31] and can assemble into dimers, trimers, tetramers, and megapores, facilitating the release of apoptogenic proteins, such as Cyto-c, apoptosis-inducing factor (AIF), and Smac/DIABLO, from the intermembrane space into the cytosol[32,33]
We examined the beneficial impact of the blockade of VDAC1 oligomerization with DIDS on initial secondary injury response, which decreased oligodendrocyte cell death, and improved axonal density, as well as allowed the motor function recovery in rats
Summary
Spinal cord injury (SCI) can affect mobility and sensibility, first mainly due to axonal rupture and secondly, due to tissue environment toxicity and the consequent inflammatory response. We were able to determine that reduced VDAC1 protein levels affected mitochondrial metabolism as observed by MTT reductase activity assay after MO treatment (54.9 ± 11.4%, P < 0.05) when compared to negative control. We performed IF using CD86 antibody, a marker of activated microglia, and we were not able to determine changes in the number of CD86-positive cells comparing the PBS and DIDS groups (Fig. 4d–f).
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