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. 2013 Feb;49(2):347-59.
doi: 10.1007/s12031-012-9873-7. Epub 2012 Aug 10.

Expression profile of flotillin-2 and its pathophysiological role after spinal cord injury

José M Santiago  1 , Aranza I Torrado, Luz C Arocho, Odrick R Rosas, Ana E Rodríguez, Franchesca König Toro, Iris K Salgado, Yaría Arroyo Torres, Walter I Silva, Jorge D Miranda
Affiliations

Expression profile of flotillin-2 and its pathophysiological role after spinal cord injury

José M Santiago et al. J Mol Neurosci. 2013 Feb.

Abstract

Some receptors that block axonal regeneration or promote cell death after spinal cord injury (SCI) are localized in membrane rafts. Flotillin-2 (Flot-2) is an essential protein associated with the formation of these domains and the clustering of membranal proteins, which may have signaling activities. Our hypothesis is that trauma will change Flot-2 expression and interference of this lipid raft marker will promote functional locomotor recovery after SCI. Analyses were conducted to determine the spatiotemporal profile of Flot-2 expression in adult rats after SCI, using the MASCIS impactor device. Immunoblots showed that SCI produced a significant decrease in the level of Flot-2 at 2 days post-injury (DPI) that increased until 28 DPI. Confocal microscopy revealed Flot-2 expression in neurons, reactive astrocytes and oligodendrocytes specifically associated to myelin structures near or close to the axons of the cord. In the open field test and grid walking assays, to monitor locomotor recovery of injured rats infused intrathecally with Flot-2 antisense oligonucleotides for 28 days showed significant behavioral improvement at 14, 21 and 28 DPI. These findings suggest that Flot-2 has a role in the nonpermissive environment that blocks locomotor recovery after SCI by clustering unfavorable proteins in membrane rafts.

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Figures

Figure 1
Figure 1
Flot-2 expression in the spinal cord and enriched in fractions characteristic of lipid raft proteins. Total homogenates of adult rat spinal cords and C6 glioma cells were evaluated by Western blot analysis using a rabbit anti-rat Flotillin-2 antibody and a mouse anti-rat Flotillin-2. The same immunoreactive bands were observed for both types of extracts when treated with both commercial antibodies (A), thus confirming the presence of Flot-2 in this tissue. Extracts from adult spinal cords were prepared with 1% Triton X-100 and the insoluble (Ins) and soluble (Sol) fractions were analyzed by Western blot analysis (B). Flot-2 was exclusively expressed in the insoluble fraction, thus demonstrating its insolubility in Triton X-100, a fundamental characteristic of lipid raft proteins. Flot-2 co-localizes with Cav-1, a lipid raft marker, in a sucrose density gradient (C). Spinal cord homogenates were studied by sucrose density gradient and Western blot analysis. Flot-2 was observed in fractions 6, 7, 8, and 9. Cav-1 also co-localized in the latter fractions, thus confirming that Flot-2 is associated with lipid rafts.
Figure 2
Figure 2
Flot-2 expression in different areas of the spinal cord of the adult rat. Immunoblot studies demonstrated the relative levels of Flot-2 expression in different areas of the spinal cord. The segments studied were cervical (C), thoracic (T), lumbar (L) and sacral (S). An ANOVA confirmed that the levels of Flot-2 expression significantly and progressively decreased along the distance of the spinal cord, with the highest levels of Flot-2 expressed in cervical region and lower levels expressed in the sacral region (p < 0.001). Data were analyzed by densitometry and are presented as mean ± SEM (n= 3–4).
Figure 3
Figure 3
Flot-2 expression profile after spinal cord injury. Flot-2 expression was altered after SCI (A). An initial decrease in this protein was observed in the acute phase after trauma. Flot-2 expression returned to basal expression at 14 DPI, and was upregulated at 28 DPI relative to GAPDH levels at these time points (B). The results of and Bonferroni post-hoc test established significant difference between the samples tested. All of the values presented are the mean ± SEM (n= 6 – 9 per group and 42 in the control group) **p < 0.01.
Figure 4
Figure 4
Flotillin-2 expression decreased in motorneurons, oligodendrocytes and axons but increased in reactive astrocytes of the injured spinal cord at 2 DPI. Immunohistochemical studies from the spinal cords of sham (A–C, G–I, M–O and S–U) and injured animals (D–F, J–L, P–R, and V–X) were performed to assess changes in Flot-2 expression (A, D, G, J, M, P, S & V in red color) in motorneurons (B & E, in green color), oligodendrocyte myelin structures (H & K in green color) axons (N & Q, in green color) and reactive astrocytes (T & W) after injury. A decrease in the number of Flot-2 immunoreactive neurons was observed in the injured rats (F, in yellow) when compared with sham rats (C in yellow). The same reduced pattern of expression was observed in myelin structures of oligodendrocytes (L vs. I) and axonal structures (R vs. O). As for the reactive astrocytes, Flot-2 immunoreactivity was observed to increase in injured animals when compared to sham animals (X vs. U). Arrows point to myelin structures of oligodendrocytes. Scale bar= 10 μm.
Figure 5
Figure 5
Blockade of Flotillin-2 facilitates functional locomotor recovery and improves fine motor movement after injury. (A) Flot-2 antisense infusion (2.5 and 5 nmoles) for 28 days after injury reduced the levels of Flot-2 as compared with the rats that were injured and treated with the scrambled control oligonucleotides. All of the values presented are the mean ± SEM (n= 3)*p < 0.05. (B) Rats treated with Flot-2 antisense showed significant improvements in functional locomotor recovery in the BBB open field test after injury as compared with the control (scramble) group, at 7, 14, 21 and 28 DPI. (C) In the grid walking test, rats treated with the Flot-2 antisense showed significantly fewer errors as compared with the control (scramble) group at 14, 21 and 28 DPI. All of the values presented are the mean ± SEM (n= 4–10) ***p < 0.001, **p < 0.01, *p < 0.05.
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