We found that adult retinal axons have a lower ratio of GM1/GD1a gangliosides on their surface than DRG axons

We found that adult retinal axons have a lower ratio of GM1/GD1a gangliosides on their surface than DRG axons. absence of eCa2+ on Neu3 sialidase activity and axonal regeneration, cultures were incubated with normal growth media devoid of CaCl2, 30 min before axotomy. siRNA transfection Dissociated DRGs were transfected with siRNA 48 h after plating. The Neu3 siRNA consists of four siRNA sequences: GCAGAGAUGCGUACCUCAA, CCAACAACUCUGCGAGCCU, CCAAACAAAUUCCGAGCAG, and GGACAGGGCUUGUUCGCGU (ON-TARGETplus SMART pool rat Neu3 siRNA, 117185, Thermo Scientific). siGLO RISC-Free siRNA (Thermo Scientific) was used as a negative control and transfection indicator. Briefly, DharmaFECT 3 (Thermo Scientific) and siRNA were SR9011 diluted separately in serum-free DMEM supplemented with 1% insulin-transferrin-selenium and 10 ng/ml NGF before mixing together and incubating with the DRGs (100 nm; 4 h) and then replacing with normal medium. RNA purification and RT-PCR Dissociated DRGs were lysed and collected as per the manufacturer’s instructions (PureLink RNA Mini Kit, Invitrogen) before being homogenized (Ultra-Turrax T8 homogenizer, IKA-Werke). RNA purification was performed before reverse transcription. The purified RNA was then reverse-transcribed Rabbit Polyclonal to POLE4 using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). The cDNA synthesis reaction was followed by PCR with the following primers: Neu3, forward, 5-CAGCTGGGATAGCAGAGGTC-3; reverse, 5-GAGTCCTGAAGCAAGCCAAC-3, resulting in a 209 bp fragment; Neu4, forward, 5-CCTGACCCTAGGACGAACAG-3; reverse, 5-GATGTGCGTGGTGATCAGAG-3, resulting in a 179 bp fragment. The PCR was run at 26, 29, and 32 cycles separately and imaged using agarose gel electrophoresis. Immunocytochemical staining of cultures Both DRG and retina explant cultures were fixed with 4% PFA for 10 min and washed with PBS before immunostaining. To analyze Neu3 sialidase activity, we calculated the relative levels of GD1a (substrate) and GM1 (product) on individual axonal membranes rather than absolute mean values of GD1a and GM1 ganglioside. A mean of 30C60 axons per chamber (2 DRGs/chamber) was considered as = 1/experimental condition. Fixed cultures were incubated with one of the following antibodies: rabbit anti-Neu3 sialidase/PMGS (Rodriguez et al., 2001), mouse anti-GD1a ganglioside (kind gift from Prof. Ronald Schnaar, Johns Hopkins University, Baltimore, MD), mouse anti-GD1a ganglioside and mouse anti-GT1b ganglioside (Merck Millipore) or stained using recombinant cholera toxin-B subunit (CTB) conjugated to AlexaFluor-555 (Invitrogen), and mouse anti 3-tubulin. To analyze the signaling cascade regulating Neu3 sialidase activity, DRG and retinal cultures were analyzed with the following primary antibodies: mouse anti-P38MAPK, rabbit anti-phospho P38MAPK, and rabbit anti-ERK and mouse anti-phospho ERK (Cell Signaling Technology). Subsequently, cultures were washed in PBS and SR9011 incubated with the following fluorescent secondary antibodies: anti-rabbit AlexaFluor-660, and anti-mouse or anti-rabbit AlexaFluor-488 (Invitrogen) before being mounted onto slides using Fluorosave (Calbiochem). Surgeries All surgeries were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and United Kingdom Home Office regulations. Adult (2- to 3-month-old) male Sprague Dawley rats were used for all experiments. Animals were kept in 12 h light/dark exposure, and food and water was provided values of all data were above set levels of 0. 05 and therefore considered to be normally distributed. Results from the analyses were expressed as mean SEM. Statistical analysis was performed using Prism version 5 (Graphpad). For all SR9011 SR9011 experiments, Student’s test (two-tailed), one-way ANOVA, and two-way ANOVA were applied as appropriate. Significant interactions were analyzed using paired tests and Bonferroni tests as appropriate. Results Axotomy leads to an increase in the ratio of GM1/GD1a gangliosides due to Neu3 sialidase activation As a model of the first steps in successful axon regeneration, we used regeneration of growth cones in adult DRG axons grown on PDL/laminin. These axons regenerate a new growth cone and begin to elongate within 30 min after axotomy (Chierzi et al., 2005). We investigated levels of GM1 and GD1a gangliosides.