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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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Modeling of autism-associated NHE9 variants(A) Top and side views of a model-structure of the membrane domain of NHE9 based on the structure of E. coli NhaA and colored according to degree of ConSurf conservation, with turquoise through maroon indicating variable through conserved amino acid positions. Three autism-associated variants (S438P, L236S, V176I) are shown in space-filled form. (B) Site-directed mutagenesis was used to introduce equivalent NHE9 mutations into yeast Nhx1 (A438P, I222S, and V167I) as well as ‘humanized’ variants A438S and I222L to mimic wild type NHE9. (C) Nhx1 constructs tagged with GFP were expressed in the nhx1Δ  strain and visualized (100× objective) as fluorescent punctae, characteristic of pre-vacuolar compartments. Scale bar: 20 µm (D) Immunoblot, with anti-HA, was used to detect similar expression levels of HA-tagged Nhx1 and variants. GAPDH was used as loading control.
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Figure 2: Modeling of autism-associated NHE9 variants(A) Top and side views of a model-structure of the membrane domain of NHE9 based on the structure of E. coli NhaA and colored according to degree of ConSurf conservation, with turquoise through maroon indicating variable through conserved amino acid positions. Three autism-associated variants (S438P, L236S, V176I) are shown in space-filled form. (B) Site-directed mutagenesis was used to introduce equivalent NHE9 mutations into yeast Nhx1 (A438P, I222S, and V167I) as well as ‘humanized’ variants A438S and I222L to mimic wild type NHE9. (C) Nhx1 constructs tagged with GFP were expressed in the nhx1Δ strain and visualized (100× objective) as fluorescent punctae, characteristic of pre-vacuolar compartments. Scale bar: 20 µm (D) Immunoblot, with anti-HA, was used to detect similar expression levels of HA-tagged Nhx1 and variants. GAPDH was used as loading control.

Mentions: As a first step in determining whether rare coding variants in NHE9 contributed to the autism phenotype, we constructed a structural model of the membrane domain of NHE9, and its yeast ortholog Nhx1, based on the crystal structure of a distantly related bacterial ortholog, NhaA35. Previously, we used evolutionary analysis and a composite fold-recognition approach to propose a three-dimensional model-structure of NHE1, a prototype of the cation/proton superfamily36,37. Utilizing a similar methodology, we aligned yeast Nhx1 and mammalian NHE9 to NhaA (Figure 1A), as well as to NHE1. In accordance with phylogenetic clustering, the resulting alignments showed that both Nhx1 and NHE9 were significantly more closely related to NHE1 than to bacterial NhaA, with sequence identities of 30% and 32% for the alignments of Nhx1 and NHE9 to NHE1, respectively, whereas aligning Nhx1 and NHE9 to NhaA resulted in sequence identities of 15% and 14%, respectively. This allowed us to extend the structural model from NHE1 to NHE9 (Figure 1B and Figure 2A) and Nhx1 (Figure 1C). To evaluate the Nhx1 and NHE9 models, we examined characteristic traits of membrane proteins, namely the distribution of hydrophobicity and evolutionary conservation. Consistent with the reliability of the models, we show a preponderance of hydrophobic residues within the predicted membrane spans (Figure 1B), and concentration of evolutionarily conserved residues within the core regions of the transporter (Figure 2A).


Functional evaluation of autism-associated mutations in NHE9.

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

Modeling of autism-associated NHE9 variants(A) Top and side views of a model-structure of the membrane domain of NHE9 based on the structure of E. coli NhaA and colored according to degree of ConSurf conservation, with turquoise through maroon indicating variable through conserved amino acid positions. Three autism-associated variants (S438P, L236S, V176I) are shown in space-filled form. (B) Site-directed mutagenesis was used to introduce equivalent NHE9 mutations into yeast Nhx1 (A438P, I222S, and V167I) as well as ‘humanized’ variants A438S and I222L to mimic wild type NHE9. (C) Nhx1 constructs tagged with GFP were expressed in the nhx1Δ  strain and visualized (100× objective) as fluorescent punctae, characteristic of pre-vacuolar compartments. Scale bar: 20 µm (D) Immunoblot, with anti-HA, was used to detect similar expression levels of HA-tagged Nhx1 and variants. GAPDH was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815575&req=5

Figure 2: Modeling of autism-associated NHE9 variants(A) Top and side views of a model-structure of the membrane domain of NHE9 based on the structure of E. coli NhaA and colored according to degree of ConSurf conservation, with turquoise through maroon indicating variable through conserved amino acid positions. Three autism-associated variants (S438P, L236S, V176I) are shown in space-filled form. (B) Site-directed mutagenesis was used to introduce equivalent NHE9 mutations into yeast Nhx1 (A438P, I222S, and V167I) as well as ‘humanized’ variants A438S and I222L to mimic wild type NHE9. (C) Nhx1 constructs tagged with GFP were expressed in the nhx1Δ strain and visualized (100× objective) as fluorescent punctae, characteristic of pre-vacuolar compartments. Scale bar: 20 µm (D) Immunoblot, with anti-HA, was used to detect similar expression levels of HA-tagged Nhx1 and variants. GAPDH was used as loading control.
Mentions: As a first step in determining whether rare coding variants in NHE9 contributed to the autism phenotype, we constructed a structural model of the membrane domain of NHE9, and its yeast ortholog Nhx1, based on the crystal structure of a distantly related bacterial ortholog, NhaA35. Previously, we used evolutionary analysis and a composite fold-recognition approach to propose a three-dimensional model-structure of NHE1, a prototype of the cation/proton superfamily36,37. Utilizing a similar methodology, we aligned yeast Nhx1 and mammalian NHE9 to NhaA (Figure 1A), as well as to NHE1. In accordance with phylogenetic clustering, the resulting alignments showed that both Nhx1 and NHE9 were significantly more closely related to NHE1 than to bacterial NhaA, with sequence identities of 30% and 32% for the alignments of Nhx1 and NHE9 to NHE1, respectively, whereas aligning Nhx1 and NHE9 to NhaA resulted in sequence identities of 15% and 14%, respectively. This allowed us to extend the structural model from NHE1 to NHE9 (Figure 1B and Figure 2A) and Nhx1 (Figure 1C). To evaluate the Nhx1 and NHE9 models, we examined characteristic traits of membrane proteins, namely the distribution of hydrophobicity and evolutionary conservation. Consistent with the reliability of the models, we show a preponderance of hydrophobic residues within the predicted membrane spans (Figure 1B), and concentration of evolutionarily conserved residues within the core regions of the transporter (Figure 2A).

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