PMC

Drivers of neuronal morphology affected by G-CSF. IPA analysis of the most significantly altered cellular functions following G-CSF treatment revealed that proteins involved in altering neuronal morphology were significantly changed. This diagram shows all significantly regulated proteins predicted to be involved in affecting neuronal morphology, and their corresponding predicted subcellular distribution. Proteins visualized in red are significantly increased, and those in green were significantly decreased.

Comparison of significantly-regulated proteins between all treatment groups. (A) Venn diagram demonstrating overlap and differences of proteins changed between the three treatment groups relative to saline controls. (B) Heatmap visualization of the 789 proteins that were significantly regulated in any treatment group demonstrates clusters of proteins that are differentially affected based on the three treatment groups. K-means clustering (k = 5) used to create heatmap of z-scored mean fold-change from saline.

Key upstream regulators of proteins altered by G-CSF. From the proteins identified as significantly altered by repeated G-CSF treatment, we used IPA analysis to identify key upstream regulators. Two of the most robust were FMRP (A) and mTOR (B). These dendrograms represent all proteins that were significantly changed in this dataset that are predicted to be directly downstream of these regulators, and those that are predicted to be directly downstream of those (two degrees of regulation). Proteins visualized in red are significantly increased, and those in green were significantly decreased.

Western blot validations—To validate proteins identified as changed by mass spectrometry additional Western blot analysis of similarly treated tissue was performed. Graphical fold-change from saline control is shown for tyrosine hydroxylase (A) and Mecp2 (B) with corresponding representative images shown below. Blue lines on the graph represent the fold-change from saline that was seen in each of the treatment groups with mass spec analysis.

Changes in VTA protein expression in cocaine-treated groups. Volcano plots of proteins in the Cocaine (A) and Cocaine + G-CSF (B) groups relative to saline controls. Log2 Fold change on the x-axis and Log10 p value on the y-axis. Proteins that were significantly changed with a nominal p value of <0.05 are represented by blue dots, and those with a ±20% change and a p < 0.05 are represented by green dots. (C) Demonstrates the changes in the Cocaine + G-CSF group relative to the Cocaine group. Proteins with a nominal p value of <0.05 are represented by turquoise dots, and those with a ± 20% change and a p < 0.05 are represented by red dots.

G-CSF regulated proteins and signaling pathways. (A) Volcano plot demonstrating proteins in the G-CSF group relative to saline controls with Log2 Fold change on the x-axis and Log10 p value on the y-axis. Proteins that were significantly changed with a nominal p value of <0.05 are represented by blue dots, and those with a ±20% change and a p < 0.05 are represented by green dots. Ingenuity pathway analysis demonstrated that amongst the significantly regulated proteins there were multiple canonical signaling pathways that were found to be down-regulated (B) as well as up-regulated (C) relative to saline controls.

Canonical pathways regulated in all groups and predicted transcription factors of upregulated proteins. (A) Ingenuity Pathway Analysis software was used to compare significantly altered canonical signaling pathways amongst all treatment groups. The height of the bars represents the statistical strength of the change but does not represent directionality of change. Directional data available in . (B) Using Enrichr software we identified transcription factors with the highest predicted number of targets in our datasets. This graph demonstrates the calculated transcription factor (TF) prediction score for the three chosen transcription factors. Significantly regulated targets are available in .

Experimental design and validation. (A) Animals were injected with ± G-CSF (50 μg/kg) ± Cocaine (7.5 mg/kg) a 2 × 2 design. Injections were done once daily for 7 days and animals sacrificed 24 h after the final injection and the VTA dissected out for analysis. (B) To allow for significant power, two runs of the TMT 10-plex were run with two samples from each group per run (total 4/group) with a mix comprised of an equal amount of each sample run as a Master Mix run to allow normalization between runs. Median intensity values of actin, which was not significantly changed in any group show near complete overlap (C) whereas Mecp2 shows increase in all non-saline groups with the expected change in median intensity (D).