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Functional evaluation of autism-associated mutations in NHE9.

Kondapalli KC, Hack A, Schushan M, Landau M, Ben-Tal N, Rao R - Nat Commun (2013)

Bottom Line: Here we use evolutionary conservation analysis to build a model structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse.NHE9-GFP localizes to recycling endosomes, where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter.In contrast, autism-associated variants L236S, S438P and V176I lack function in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA [2].

ABSTRACT
NHE9 (SLC9A9) is an endosomal cation/proton antiporter with orthologues in yeast and bacteria. Rare, missense substitutions in NHE9 are genetically linked with autism but have not been functionally evaluated. Here we use evolutionary conservation analysis to build a model structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse. NHE9-GFP localizes to recycling endosomes, where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter. In contrast, autism-associated variants L236S, S438P and V176I lack function in astrocytes. Thus, we establish a neurobiological cell model of a candidate gene in autism. Loss-of-function mutations in NHE9 may contribute to autistic phenotype by modulating synaptic membrane protein expression and neurotransmitter clearance.

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NHE9 regulates endosomal pH(A) Calibration of endosomal pH from fluorescence ratio of internalized Tf-FITC and Tf-Alexafluor. Cells were loaded with tagged Transferrin (Tf) for 1 hr, then exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0). Internalized Tf was quantified using flow cytometry. (B) NHE9 expression alkalinizes endosomal lumen. pH of Tfn-positive endosomes in primary cultured cortical astrocytes was determined in control, NHE9 overexpression, and NHE9 shRNA knock-down conditions. Results are averages of three biological replicates, each done in triplicate (* p < 0.05). (C) Knockdown of NHE9 acidifies Tfn-positive endosomes in primary cultured human glioma cells. Results are averages of three replicates (* p < 0.05). Statistical analysis was by Student’s t test; all error bars represent SD.
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Figure 6: NHE9 regulates endosomal pH(A) Calibration of endosomal pH from fluorescence ratio of internalized Tf-FITC and Tf-Alexafluor. Cells were loaded with tagged Transferrin (Tf) for 1 hr, then exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0). Internalized Tf was quantified using flow cytometry. (B) NHE9 expression alkalinizes endosomal lumen. pH of Tfn-positive endosomes in primary cultured cortical astrocytes was determined in control, NHE9 overexpression, and NHE9 shRNA knock-down conditions. Results are averages of three biological replicates, each done in triplicate (* p < 0.05). (C) Knockdown of NHE9 acidifies Tfn-positive endosomes in primary cultured human glioma cells. Results are averages of three replicates (* p < 0.05). Statistical analysis was by Student’s t test; all error bars represent SD.

Mentions: To investigate the effect of altered NHE9 levels on lumenal pH in recycling endosomes, we took advantage of the excellent overlap in localization with transferrin (Figure 7B, top panel), following 60 min uptake into live cells. Fluorescence ratio imaging was done by using a combination of pH-sensitive FITC-tagged Transferrin with pH-insensitive Alexa Fluor-tagged Transferrin as control for transferrin loading, and the endosomal pH was determined from a calibration curve (Figure 6A). Relative to the control cells (pH 5.7 ± 0.22), endosomal pH in NHE9 overexpressing cells was more alkaline (pH 6.39 ± 0.054), as expected from Na+(K+)/H+ exchange mediating proton leak from the endosomes (Figure 6B). These results are consistent with increased endosomal pH observed by Nakamura et al. in COS7 cells overexpressing NHE98. Although luminal pH decreased upon knockdown of NHE9 (to pH 5.39, p=0.08; Student's t-test, n= three biological replicates; Figure 6B), the difference fell short of significance. Therefore, we examined the effect of NHE9 knockdown in primary cultured human glioma cells (Figure 6C). We did observe significant acidification of endosomes upon NHE9 knockdown (pH 6.60, p<0.05; Student's t-test, n= three biological replicates) relative to control (pH 6.88). These results suggest functional differences between mouse and human astrocyte cells, consistent with the limitations of the mouse model in recapitulating human disease. It is possible that NHE6 compensates for loss of NHE9 in mouse cortical astrocytes. Indeed, we observed high levels of co-localization of NHE6-GFP and NHE9-DsRed in murine astrocytes (Figure 5E–F), consistent with redundant roles for NHE6 and NHE9 in regulating endosomal pH.


Functional evaluation of autism-associated mutations in NHE9.

Kondapalli KC, Hack A, Schushan M, Landau M, Ben-Tal N, Rao R - Nat Commun (2013)

NHE9 regulates endosomal pH(A) Calibration of endosomal pH from fluorescence ratio of internalized Tf-FITC and Tf-Alexafluor. Cells were loaded with tagged Transferrin (Tf) for 1 hr, then exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0). Internalized Tf was quantified using flow cytometry. (B) NHE9 expression alkalinizes endosomal lumen. pH of Tfn-positive endosomes in primary cultured cortical astrocytes was determined in control, NHE9 overexpression, and NHE9 shRNA knock-down conditions. Results are averages of three biological replicates, each done in triplicate (* p < 0.05). (C) Knockdown of NHE9 acidifies Tfn-positive endosomes in primary cultured human glioma cells. Results are averages of three replicates (* p < 0.05). Statistical analysis was by Student’s t test; all error bars represent SD.
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Figure 6: NHE9 regulates endosomal pH(A) Calibration of endosomal pH from fluorescence ratio of internalized Tf-FITC and Tf-Alexafluor. Cells were loaded with tagged Transferrin (Tf) for 1 hr, then exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0). Internalized Tf was quantified using flow cytometry. (B) NHE9 expression alkalinizes endosomal lumen. pH of Tfn-positive endosomes in primary cultured cortical astrocytes was determined in control, NHE9 overexpression, and NHE9 shRNA knock-down conditions. Results are averages of three biological replicates, each done in triplicate (* p < 0.05). (C) Knockdown of NHE9 acidifies Tfn-positive endosomes in primary cultured human glioma cells. Results are averages of three replicates (* p < 0.05). Statistical analysis was by Student’s t test; all error bars represent SD.
Mentions: To investigate the effect of altered NHE9 levels on lumenal pH in recycling endosomes, we took advantage of the excellent overlap in localization with transferrin (Figure 7B, top panel), following 60 min uptake into live cells. Fluorescence ratio imaging was done by using a combination of pH-sensitive FITC-tagged Transferrin with pH-insensitive Alexa Fluor-tagged Transferrin as control for transferrin loading, and the endosomal pH was determined from a calibration curve (Figure 6A). Relative to the control cells (pH 5.7 ± 0.22), endosomal pH in NHE9 overexpressing cells was more alkaline (pH 6.39 ± 0.054), as expected from Na+(K+)/H+ exchange mediating proton leak from the endosomes (Figure 6B). These results are consistent with increased endosomal pH observed by Nakamura et al. in COS7 cells overexpressing NHE98. Although luminal pH decreased upon knockdown of NHE9 (to pH 5.39, p=0.08; Student's t-test, n= three biological replicates; Figure 6B), the difference fell short of significance. Therefore, we examined the effect of NHE9 knockdown in primary cultured human glioma cells (Figure 6C). We did observe significant acidification of endosomes upon NHE9 knockdown (pH 6.60, p<0.05; Student's t-test, n= three biological replicates) relative to control (pH 6.88). These results suggest functional differences between mouse and human astrocyte cells, consistent with the limitations of the mouse model in recapitulating human disease. It is possible that NHE6 compensates for loss of NHE9 in mouse cortical astrocytes. Indeed, we observed high levels of co-localization of NHE6-GFP and NHE9-DsRed in murine astrocytes (Figure 5E–F), consistent with redundant roles for NHE6 and NHE9 in regulating endosomal pH.

Bottom Line: Here we use evolutionary conservation analysis to build a model structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse.NHE9-GFP localizes to recycling endosomes, where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter.In contrast, autism-associated variants L236S, S438P and V176I lack function in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA [2].

ABSTRACT
NHE9 (SLC9A9) is an endosomal cation/proton antiporter with orthologues in yeast and bacteria. Rare, missense substitutions in NHE9 are genetically linked with autism but have not been functionally evaluated. Here we use evolutionary conservation analysis to build a model structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse. NHE9-GFP localizes to recycling endosomes, where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter. In contrast, autism-associated variants L236S, S438P and V176I lack function in astrocytes. Thus, we establish a neurobiological cell model of a candidate gene in autism. Loss-of-function mutations in NHE9 may contribute to autistic phenotype by modulating synaptic membrane protein expression and neurotransmitter clearance.

Show MeSH
Related in: MedlinePlus