TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression

Abstract

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.

Fig. 1

SDS-PAGE analysis of GFP and GFP-TAT purity. Samples were run on a 4–12% NuPAGE Bis-Tris gel under denaturing conditions. The gel was stained with SimplyBlue Safe Stain and shows apparent homogeneity of the expressed products. The construct molecular weights compare well to the calculated theoretical molecular weights of 31,113 Da and 33,144 Da for GFP and GFP-TAT, respectively.

Fig. 2

Transduction of GFP and GFP-TAT into differentiated and undifferentiated PC12 cells. Undifferentiated PC12 cells and differentiated (NGF-treated) PC12 cells were incubated in 100 µg/mL GFP or GFP-TAT for 4 hours, and transduction was quantified using flow cytometry. Results are expressed as the percent increase in geometric mean fluorescence compared to untreated control cells, to account for background autofluorescence (n>3, error bars: ±SEM). TAT significantly enhanced GFP transduction into both undifferentiated and differentiated PC12 cells compared to GFP alone. *Significance from Student’s t-test (p<0.05). Overall GFP-TAT transduction was significantly greater in differentiated cells. ^#Significance from Student’s t-test (p<0.05).

Fig. 3

Transduction of GFP and GFP-TAT into astrocytes. Astrocytes in monoculture, grown either in serum-containing medium or serum-free Neurobasal medium, and astrocytes in coculture with neurons were incubated in 100 µg/mL GFP or GFP-TAT for 4 hours, and transduction was quantified using flow cytometry. Cell type was confirmed by GFAP immunofluorescence after trypsinization. The percent increase in geometric mean fluorescence compared to untreated control cells was calculated, to account for background autofluorescence (n>6, error bars: ±SEM). TAT significantly enhanced GFP transduction into all three astrocyte cultures compared to GFP. *Significance from Student’s t-test (p<0.05). Overall GFP-TAT transduction into cocultures was significantly less than into astrocyte monocultures grown in serum medium and serum-free medium. ^#Significance from Bonferroni post-hoc test (p<0.05).

Fig. 4

Transduction of GFP and GFP-TAT into neurons. Neurons in cocultures were incubated in 100 µg/mL GFP or GFP-TAT for 4 hours, and transduction was quantified using flow cytometry. Cell type was confirmed by Thy1 immunofluorescence after trypsinization. The percent increase in geometric mean fluorescence of Thy1+ cells compared to untreated control cells was calculated, to account for background autofluorescence (n>6, error bars: ±SEM). TAT did not significantly enhance GFP transduction into neurons, compared to GFP alone.

Fig. 5

Dependence of GFP-TAT transduction on PC12 cell GAG content. (A) The average µg of GAG / ng of DNA, normalized to the average µg of GAG / ng of DNA in undifferentiated cultures is shown for undifferentiated PC12 cells treated to experimentally alter GAG content (n>4, error bars: ±SEM). There was a significant increase in GAG with differentiation, and a significant decrease in GAG with trypsin treatment. (B) Geometric mean fluorescence of GFP-TAT was normalized to undifferentiated PC12s (n>4, error bars: ±SEM). GFP-TAT transduction significantly increased in NGF-treated PC12 cells, and significantly decreased in both trypsin and heparin treated cells. *Significance from Dunnett post-hoc comparison to the control, undifferentiated condition. (C) Percent increase in GFP-TAT transduction, compared to GFP alone, showed a positive correlation with GAG content (n>4, three data points correspond to trypsin-treated, untreated, and NGF-treated PC12 cells, error bars: ±SEM), indicating that GAG content influences the ability of TAT to enhance GFP transduction.

Fig. 6

Dependence of GFP-TAT transduction on astrocyte GAG content. (A) The average µg of GAG / ng of DNA, normalized to the average µg of GAG / ng of DNA in serum monocultures is shown for serum-grown astrocyte monocultures treated to experimentally alter GAG content (n>4, error bars: ±SEM). There was a significant increase in GAG content with ascorbic acid treatment. Trypsin treatment decreased GAG content. (B) GFP-TAT transduction was quantified in astrocyte monocultures grown in serum-containing medium as well as in monocultures treated with ascorbic acid, trypsin, or heparin. Geometric mean fluorescence was normalized to astrocyte monocultures grown in serum (n>4, error bars: ±SEM). GFP-TAT transduction was significantly increased in ascorbic acid treated astrocytes, and significantly decreased in both trypsin and heparin treated cells. (C) Modifying the growth environment of astrocytes altered the cellular GAG content. Both removal of serum and coculture with neurons resulted in a decrease in GAG. *Significance from Dunnett post-hoc comparison to the control, undifferentiated condition. (D) Percent increase in GFP-TAT transduction, compared to GFP alone, showed a positive correlation with GAG content in astrocyte monocultures grown in serum that were experimentally treated to modulate GAG (●, three data points correspond to trypsin-treated, untreated and ascorbic acid-treated astrocytes grown in serum-containing medium). Both the astrocyte monocultures grown in serum-free medium (▽) and cocultures (□) fell within the confidence bounds of the regression formed by the monocultures grown in serum, indicating that the ability of TAT to increase transduction of GFP in astrocytes correlated with GAG content.