Limits...
Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides.

Moutal A, François-Moutal L, Brittain JM, Khanna M, Khanna R - Front Cell Neurosci (2015)

Bottom Line: When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective.Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3.Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, University of Arizona Tucson, AZ, USA.

ABSTRACT
The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2) is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3) conjugated to the HIV transactivator of transcription (TAT) protein's cationic cell penetrating peptide (CPP) motif protected neurons in the face of toxic levels of Ca(2+) influx leaked in via N-methyl-D-aspartate receptor (NMDAR) hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9)), hydrophobic (membrane transport sequence (MTS) of k-fibroblast growth factor) or amphipathic (model amphipathic peptide (MAP)) CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs) derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA)-evoked Ca(2+) influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca(2+) influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (>24 h) treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

No MeSH data available.


Related in: MedlinePlus

Differential uptake and efflux of CBD3 peptides conjugated to cationic and amphipathic CPPs in cortical neurons. Cortical neurons plated onto poly-D-lysine-coated 96-well plates were incubated with FITC-labeled CBD3 peptides (10 µM) for 10 min at 37°C, washed extensively with minimal essential media without phenol red, and fluorescence was measured using a fluorescent plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm. (A) Mean fluorescence uptake of peptides into cortical neurons, normalized to the amount of protein per well, was similar between untreated and Glu/Gly challenged neurons for all peptides except TAT-CBD3, which was decreased in Glu/Gly challenged neurons compared to untreated neurons (*p < 0.01). Mean fluorescence efflux of peptides from untreated (B) or Glu/Gly-challenged (C) cortical neurons, normalized to the amount of protein per well, was significantly lower for MTS- and MAP-CBD3 at 10, 30 and 60 min compared to either TAT- or R9-CBD3 (*p < 0.01). Some error bars are smaller than the symbols. (D) Area under the curve (AUC) analyses reflecting cumulative efflux of the peptides in the indicated conditions. *p < 0.01 comparing AUC for TAT- or R9-CBD-treated control neurons vs. their respective Glu/Gly-challenged conditions. #p < 0.01 comparing AUC from stimulated cells with TAT-CBD3 vs. R9-CBD3-treated. n = 2 separate, individual experiments; the total number of wells analyzed is 8–13 per condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4306314&req=5

Figure 2: Differential uptake and efflux of CBD3 peptides conjugated to cationic and amphipathic CPPs in cortical neurons. Cortical neurons plated onto poly-D-lysine-coated 96-well plates were incubated with FITC-labeled CBD3 peptides (10 µM) for 10 min at 37°C, washed extensively with minimal essential media without phenol red, and fluorescence was measured using a fluorescent plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm. (A) Mean fluorescence uptake of peptides into cortical neurons, normalized to the amount of protein per well, was similar between untreated and Glu/Gly challenged neurons for all peptides except TAT-CBD3, which was decreased in Glu/Gly challenged neurons compared to untreated neurons (*p < 0.01). Mean fluorescence efflux of peptides from untreated (B) or Glu/Gly-challenged (C) cortical neurons, normalized to the amount of protein per well, was significantly lower for MTS- and MAP-CBD3 at 10, 30 and 60 min compared to either TAT- or R9-CBD3 (*p < 0.01). Some error bars are smaller than the symbols. (D) Area under the curve (AUC) analyses reflecting cumulative efflux of the peptides in the indicated conditions. *p < 0.01 comparing AUC for TAT- or R9-CBD-treated control neurons vs. their respective Glu/Gly-challenged conditions. #p < 0.01 comparing AUC from stimulated cells with TAT-CBD3 vs. R9-CBD3-treated. n = 2 separate, individual experiments; the total number of wells analyzed is 8–13 per condition.

Mentions: The varying propensities of the CBD3-conjugated peptides to segregate into lipid domains may contribute to their accumulation into and efflux from cortical neuron membranes. Therefore, we next quantitatively tested influx and rates of efflux of the peptides in cortical neurons without or following an excitotoxic challenge. FITC fluorescence was measured in cortical neurons following incubation with 20 µM fluorescently labeled CBD3 peptides. To minimize any possible variability in uptake due to differences in cell plating, we normalized the fluorescence per well to the amount of protein. The fluorescence intensities were not different between control- and Glu/Gly-treated neurons for all peptides except TAT-CBD3, which exhibited a significantly lower influx in cells challenged with glutamate toxicity (Figure 2A). Influx of MTS- and MAP-CBD3 peptides was less than that of TAT- and R9-CBD3-treated neurons irrespective of the excitotoxic challenge to the neurons (Figure 2A).


Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides.

Moutal A, François-Moutal L, Brittain JM, Khanna M, Khanna R - Front Cell Neurosci (2015)

Differential uptake and efflux of CBD3 peptides conjugated to cationic and amphipathic CPPs in cortical neurons. Cortical neurons plated onto poly-D-lysine-coated 96-well plates were incubated with FITC-labeled CBD3 peptides (10 µM) for 10 min at 37°C, washed extensively with minimal essential media without phenol red, and fluorescence was measured using a fluorescent plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm. (A) Mean fluorescence uptake of peptides into cortical neurons, normalized to the amount of protein per well, was similar between untreated and Glu/Gly challenged neurons for all peptides except TAT-CBD3, which was decreased in Glu/Gly challenged neurons compared to untreated neurons (*p < 0.01). Mean fluorescence efflux of peptides from untreated (B) or Glu/Gly-challenged (C) cortical neurons, normalized to the amount of protein per well, was significantly lower for MTS- and MAP-CBD3 at 10, 30 and 60 min compared to either TAT- or R9-CBD3 (*p < 0.01). Some error bars are smaller than the symbols. (D) Area under the curve (AUC) analyses reflecting cumulative efflux of the peptides in the indicated conditions. *p < 0.01 comparing AUC for TAT- or R9-CBD-treated control neurons vs. their respective Glu/Gly-challenged conditions. #p < 0.01 comparing AUC from stimulated cells with TAT-CBD3 vs. R9-CBD3-treated. n = 2 separate, individual experiments; the total number of wells analyzed is 8–13 per condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306314&req=5

Figure 2: Differential uptake and efflux of CBD3 peptides conjugated to cationic and amphipathic CPPs in cortical neurons. Cortical neurons plated onto poly-D-lysine-coated 96-well plates were incubated with FITC-labeled CBD3 peptides (10 µM) for 10 min at 37°C, washed extensively with minimal essential media without phenol red, and fluorescence was measured using a fluorescent plate reader at an excitation wavelength of 485 nm and emission wavelength of 520 nm. (A) Mean fluorescence uptake of peptides into cortical neurons, normalized to the amount of protein per well, was similar between untreated and Glu/Gly challenged neurons for all peptides except TAT-CBD3, which was decreased in Glu/Gly challenged neurons compared to untreated neurons (*p < 0.01). Mean fluorescence efflux of peptides from untreated (B) or Glu/Gly-challenged (C) cortical neurons, normalized to the amount of protein per well, was significantly lower for MTS- and MAP-CBD3 at 10, 30 and 60 min compared to either TAT- or R9-CBD3 (*p < 0.01). Some error bars are smaller than the symbols. (D) Area under the curve (AUC) analyses reflecting cumulative efflux of the peptides in the indicated conditions. *p < 0.01 comparing AUC for TAT- or R9-CBD-treated control neurons vs. their respective Glu/Gly-challenged conditions. #p < 0.01 comparing AUC from stimulated cells with TAT-CBD3 vs. R9-CBD3-treated. n = 2 separate, individual experiments; the total number of wells analyzed is 8–13 per condition.
Mentions: The varying propensities of the CBD3-conjugated peptides to segregate into lipid domains may contribute to their accumulation into and efflux from cortical neuron membranes. Therefore, we next quantitatively tested influx and rates of efflux of the peptides in cortical neurons without or following an excitotoxic challenge. FITC fluorescence was measured in cortical neurons following incubation with 20 µM fluorescently labeled CBD3 peptides. To minimize any possible variability in uptake due to differences in cell plating, we normalized the fluorescence per well to the amount of protein. The fluorescence intensities were not different between control- and Glu/Gly-treated neurons for all peptides except TAT-CBD3, which exhibited a significantly lower influx in cells challenged with glutamate toxicity (Figure 2A). Influx of MTS- and MAP-CBD3 peptides was less than that of TAT- and R9-CBD3-treated neurons irrespective of the excitotoxic challenge to the neurons (Figure 2A).

Bottom Line: When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective.Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3.Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, University of Arizona Tucson, AZ, USA.

ABSTRACT
The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2) is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3) conjugated to the HIV transactivator of transcription (TAT) protein's cationic cell penetrating peptide (CPP) motif protected neurons in the face of toxic levels of Ca(2+) influx leaked in via N-methyl-D-aspartate receptor (NMDAR) hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9)), hydrophobic (membrane transport sequence (MTS) of k-fibroblast growth factor) or amphipathic (model amphipathic peptide (MAP)) CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs) derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA)-evoked Ca(2+) influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca(2+) influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (>24 h) treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

No MeSH data available.


Related in: MedlinePlus