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Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

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NHE5 modulates NGF-induced neurite growth in PC12 cells. (A, B) Control PC12 cells or PC12 cells stably expressing NHE5 shRNA (shNHE5) or NHE1 shRNA (shNHE1) were treated with 50 ng/ml NGF for 72 h to induce differentiation. Cells were labeled to visualize actin (green) and tubulin (red). Scale bars, 100 μm (A), 40 μm (B). (C) The percentage of cells in the population that formed at least one neurite ≥15 μm in length. In all cases N = 1700–1800 cells; error bars represent SEM; **p < 0.01 (Student's t test) for difference from control cells. (D, E) Mean total neurite number (D) and length (E) in individual cells that had clearly differentiated (at least one neurite ≥15 μm in length). Under each condition N ≥ 80 cells.
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Figure 8: NHE5 modulates NGF-induced neurite growth in PC12 cells. (A, B) Control PC12 cells or PC12 cells stably expressing NHE5 shRNA (shNHE5) or NHE1 shRNA (shNHE1) were treated with 50 ng/ml NGF for 72 h to induce differentiation. Cells were labeled to visualize actin (green) and tubulin (red). Scale bars, 100 μm (A), 40 μm (B). (C) The percentage of cells in the population that formed at least one neurite ≥15 μm in length. In all cases N = 1700–1800 cells; error bars represent SEM; **p < 0.01 (Student's t test) for difference from control cells. (D, E) Mean total neurite number (D) and length (E) in individual cells that had clearly differentiated (at least one neurite ≥15 μm in length). Under each condition N ≥ 80 cells.

Mentions: Having shown that NHE5, a significant endosomal acidifier in PC12 cells, influences the cell-surface abundance of TrkA and the downstream signaling of NGF-TrkA, we postulated that NHE5 might modulate NGF-induced neurite outgrowth. Control PC12 cells and PC12 cells expressing shNHE5 or shNHE1 were treated with NGF (50 ng/ml) for 72 h, followed by fixation and cytochemical visualization of actin and tubulin as a reliable read-out of cellular morphology. After NGF treatment, cells were examined for the presence or absence of neurite extensions of at least 15 μm in length emanating from the cell body (Sin et al., 2009). Compared to control cells, in which nearly 50% of the cells extended neurites, only ∼20% of shNHE5 cells formed neurites (Figure 8, A and C). Moreover, the shNHE5 cells that did form neurites exhibited a significant reduction in total neurite number and length compared with the control cells (Figure 8, B, D, and E). This reduction in neurite outgrowth was seen in multiple shNHE5 clones using three independent shRNA constructs (Supplemental Figure S3). Of interest, shNHE1 cells showed a small but significant increase in the number of cells forming neurites (Figure 8, A and C), a trend opposite to that observed in shNHE5 cells. However, in shNHE1 cells that did form neurites, the mean total neurite number and length were significantly reduced (Figure 8, A–D), in agreement with our previous study showing that pharmacological inhibition or genetic ablation of NHE1 adversely affect neurite elongation and branching (Sin et al., 2009). Thus, whereas knockdown of either NHE5 or NHE1 led to reductions in neurite number and length in cells that actually formed neurites, only knockdown of NHE5 impaired the cell's ability to differentiate in response to NGF treatment, as evidenced by the reduced number of cells with any neurite formation (Figure 8C). To further investigate the specificity of the effects of shRNA, we generated double stable cells expressing both shNHE5 and hemagglutinin (HA)-tagged human NHE5 (NHE536HA), which has a sequence different from that of rat NHE5 and is resistant to shNHE5 (Diering et al., 2011). The defective neurite morphology observed in shNHE5 cells was partially rescued in the double stable cells (Figure 9, A–D). We next tested whether heterologously expressed NHE5 can rescue the defective NGF-induced signaling observed in shNHE5 cells. As shown in Figure 9, E–G, NHE5 expression significantly increased phosphorylation of Akt and Erk1/2 in response to NGF stimulation. Finally, we transiently transfected shNHE5 into rat cortical neurons in primary culture and examined the effect on neurite formation. As shown in Figure 10, A–C, both neurite number and length were slightly but significantly reduced as a result of shNHE5 expression. Although TrkA may be expressed in cultured cortical neurons, depending on culture conditions (Counts et al., 2004), TrkB is the predominant Trk receptor in cortex (Barbacid, 1994; Huang and Reichardt, 2001). Thus it will be important to test the effect of NHE5 on TrkB trafficking behavior and signaling.


Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

NHE5 modulates NGF-induced neurite growth in PC12 cells. (A, B) Control PC12 cells or PC12 cells stably expressing NHE5 shRNA (shNHE5) or NHE1 shRNA (shNHE1) were treated with 50 ng/ml NGF for 72 h to induce differentiation. Cells were labeled to visualize actin (green) and tubulin (red). Scale bars, 100 μm (A), 40 μm (B). (C) The percentage of cells in the population that formed at least one neurite ≥15 μm in length. In all cases N = 1700–1800 cells; error bars represent SEM; **p < 0.01 (Student's t test) for difference from control cells. (D, E) Mean total neurite number (D) and length (E) in individual cells that had clearly differentiated (at least one neurite ≥15 μm in length). Under each condition N ≥ 80 cells.
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Figure 8: NHE5 modulates NGF-induced neurite growth in PC12 cells. (A, B) Control PC12 cells or PC12 cells stably expressing NHE5 shRNA (shNHE5) or NHE1 shRNA (shNHE1) were treated with 50 ng/ml NGF for 72 h to induce differentiation. Cells were labeled to visualize actin (green) and tubulin (red). Scale bars, 100 μm (A), 40 μm (B). (C) The percentage of cells in the population that formed at least one neurite ≥15 μm in length. In all cases N = 1700–1800 cells; error bars represent SEM; **p < 0.01 (Student's t test) for difference from control cells. (D, E) Mean total neurite number (D) and length (E) in individual cells that had clearly differentiated (at least one neurite ≥15 μm in length). Under each condition N ≥ 80 cells.
Mentions: Having shown that NHE5, a significant endosomal acidifier in PC12 cells, influences the cell-surface abundance of TrkA and the downstream signaling of NGF-TrkA, we postulated that NHE5 might modulate NGF-induced neurite outgrowth. Control PC12 cells and PC12 cells expressing shNHE5 or shNHE1 were treated with NGF (50 ng/ml) for 72 h, followed by fixation and cytochemical visualization of actin and tubulin as a reliable read-out of cellular morphology. After NGF treatment, cells were examined for the presence or absence of neurite extensions of at least 15 μm in length emanating from the cell body (Sin et al., 2009). Compared to control cells, in which nearly 50% of the cells extended neurites, only ∼20% of shNHE5 cells formed neurites (Figure 8, A and C). Moreover, the shNHE5 cells that did form neurites exhibited a significant reduction in total neurite number and length compared with the control cells (Figure 8, B, D, and E). This reduction in neurite outgrowth was seen in multiple shNHE5 clones using three independent shRNA constructs (Supplemental Figure S3). Of interest, shNHE1 cells showed a small but significant increase in the number of cells forming neurites (Figure 8, A and C), a trend opposite to that observed in shNHE5 cells. However, in shNHE1 cells that did form neurites, the mean total neurite number and length were significantly reduced (Figure 8, A–D), in agreement with our previous study showing that pharmacological inhibition or genetic ablation of NHE1 adversely affect neurite elongation and branching (Sin et al., 2009). Thus, whereas knockdown of either NHE5 or NHE1 led to reductions in neurite number and length in cells that actually formed neurites, only knockdown of NHE5 impaired the cell's ability to differentiate in response to NGF treatment, as evidenced by the reduced number of cells with any neurite formation (Figure 8C). To further investigate the specificity of the effects of shRNA, we generated double stable cells expressing both shNHE5 and hemagglutinin (HA)-tagged human NHE5 (NHE536HA), which has a sequence different from that of rat NHE5 and is resistant to shNHE5 (Diering et al., 2011). The defective neurite morphology observed in shNHE5 cells was partially rescued in the double stable cells (Figure 9, A–D). We next tested whether heterologously expressed NHE5 can rescue the defective NGF-induced signaling observed in shNHE5 cells. As shown in Figure 9, E–G, NHE5 expression significantly increased phosphorylation of Akt and Erk1/2 in response to NGF stimulation. Finally, we transiently transfected shNHE5 into rat cortical neurons in primary culture and examined the effect on neurite formation. As shown in Figure 10, A–C, both neurite number and length were slightly but significantly reduced as a result of shNHE5 expression. Although TrkA may be expressed in cultured cortical neurons, depending on culture conditions (Counts et al., 2004), TrkB is the predominant Trk receptor in cortex (Barbacid, 1994; Huang and Reichardt, 2001). Thus it will be important to test the effect of NHE5 on TrkB trafficking behavior and signaling.

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Show MeSH
Related in: MedlinePlus