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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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Autism-associated NHE9 variants fail to alkalinize endosomal pHCells were loaded with FITC- and Alexa Fluor-tagged Transferrin (Tf) for 55 minutes and internalized Tf was quantified using flow cytometry from at least 5,000 cells, in triplicate. pH was calibrated using the ratio of internalized Tf-FITC (pH-sensitive) and Tf-Alexa Fluor (pH-insensitive). Cells were exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0) for calibration of pH dependent fluorescence. (A) Expression of NHE9, but not autism-associated variants, in HEK293 cells results in alkalinization of endosomal pH. (B) NHE9 and autism-associated variants were expressed in primary mouse astrocytes, with similar results as in (A). Error bars represent SD.
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Figure 10: Autism-associated NHE9 variants fail to alkalinize endosomal pHCells were loaded with FITC- and Alexa Fluor-tagged Transferrin (Tf) for 55 minutes and internalized Tf was quantified using flow cytometry from at least 5,000 cells, in triplicate. pH was calibrated using the ratio of internalized Tf-FITC (pH-sensitive) and Tf-Alexa Fluor (pH-insensitive). Cells were exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0) for calibration of pH dependent fluorescence. (A) Expression of NHE9, but not autism-associated variants, in HEK293 cells results in alkalinization of endosomal pH. (B) NHE9 and autism-associated variants were expressed in primary mouse astrocytes, with similar results as in (A). Error bars represent SD.

Mentions: A function specific to astrocytes at the excitatory synapse is clearance of excess glutamate. We therefore investigated the effect of NHE9 and its variants on glutamate uptake in astrocytes. GLAST (GLutamate ASpartate Transporter) is a high-affinity, Na+-dependent glutamate transporter highly expressed in astrocytes46 where it partially co-localizes with endosomal NHE9 (Figure 9A–D). Overexpression of NHE9 resulted in ~1.9 fold increase in 3H-glutamate uptake relative to control cells, whereas all three autism-associated variants were similar to vector-transformed control (Figure 9E). Although total amounts remained unchanged in all cell lines, surface expression of GLAST transporter increased by ~2 fold in astrocytes expressing wild type NHE9 but not the autism-associated variants L236S, S438P and V176I (Figure 9 F–G). Consistent with these observations, alkalinization of the transferrin-positive endosomal compartment was only observed in cells expressing wild type NHE9 (Figure 10A–B). Taken together, our findings indicate that all three autism-associated variants were associated with loss of function phenotypes in astrocytes.


Functional evaluation of autism-associated mutations in NHE9.

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

Autism-associated NHE9 variants fail to alkalinize endosomal pHCells were loaded with FITC- and Alexa Fluor-tagged Transferrin (Tf) for 55 minutes and internalized Tf was quantified using flow cytometry from at least 5,000 cells, in triplicate. pH was calibrated using the ratio of internalized Tf-FITC (pH-sensitive) and Tf-Alexa Fluor (pH-insensitive). Cells were exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0) for calibration of pH dependent fluorescence. (A) Expression of NHE9, but not autism-associated variants, in HEK293 cells results in alkalinization of endosomal pH. (B) NHE9 and autism-associated variants were expressed in primary mouse astrocytes, with similar results as in (A). Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815575&req=5

Figure 10: Autism-associated NHE9 variants fail to alkalinize endosomal pHCells were loaded with FITC- and Alexa Fluor-tagged Transferrin (Tf) for 55 minutes and internalized Tf was quantified using flow cytometry from at least 5,000 cells, in triplicate. pH was calibrated using the ratio of internalized Tf-FITC (pH-sensitive) and Tf-Alexa Fluor (pH-insensitive). Cells were exposed to nigericin (100µM) and pH defined medium (pH 5.0 to pH 8.0) for calibration of pH dependent fluorescence. (A) Expression of NHE9, but not autism-associated variants, in HEK293 cells results in alkalinization of endosomal pH. (B) NHE9 and autism-associated variants were expressed in primary mouse astrocytes, with similar results as in (A). Error bars represent SD.
Mentions: A function specific to astrocytes at the excitatory synapse is clearance of excess glutamate. We therefore investigated the effect of NHE9 and its variants on glutamate uptake in astrocytes. GLAST (GLutamate ASpartate Transporter) is a high-affinity, Na+-dependent glutamate transporter highly expressed in astrocytes46 where it partially co-localizes with endosomal NHE9 (Figure 9A–D). Overexpression of NHE9 resulted in ~1.9 fold increase in 3H-glutamate uptake relative to control cells, whereas all three autism-associated variants were similar to vector-transformed control (Figure 9E). Although total amounts remained unchanged in all cell lines, surface expression of GLAST transporter increased by ~2 fold in astrocytes expressing wild type NHE9 but not the autism-associated variants L236S, S438P and V176I (Figure 9 F–G). Consistent with these observations, alkalinization of the transferrin-positive endosomal compartment was only observed in cells expressing wild type NHE9 (Figure 10A–B). Taken together, our findings indicate that all three autism-associated variants were associated with loss of function phenotypes in astrocytes.

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