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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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Related in: MedlinePlus

Developmental regulation of NHE9 in mouse and expression in primary brain cells(A) Raw expression levels of NHE9 in various regions of the mouse brain determined from in situ hybridization data obtained from Allen Brain Atlas [Available from: http://mouse.brain-map.org/]. ISOCTX: Isocortex, OLF: Olfactory areas, HPF: Hippocampal formation, CTXsp: Cortical subplate, STR: Striatum, PAL: Pallidum, CB: Cerebellum, TH: Thalamus, HY: Hypothalamus, MB: Midbrain, P: Pons, MY: Medulla. (B) NHE9 gene expression in developing mouse brain characterized by in situ hybridization (ISH) in sagittal plane across four embryonic and three early postnatal ages. Feulgen-HP yellow DNA counterstain, a nuclear stain, was used to add definition to the tissue. This counterstain is used in conjunction with ISH for all data shown except for P56, in order to provide tissue context to the ISH signal which is otherwise difficult to discern due to the very light tissue background for embryonic ISH. Images were obtained from Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org] (C)In situ hybridization data showing expression summary of NHE9 in the various regions of the mouse brain during development, obtained from Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org]. RSP: Rostral secondary prosencephalon, Tel: Telencephalic vesicle, PHy: Peduncular (caudal) hypothalamus, p3: Prosomere 3, p2: Prosomere 2, p1: Prosomere 1, M: Midbrain, PPH: Prepontine hindbrain, PH: Pontine hindbrain, PMH: Pontomedullary hindbrain, MH: Medullary hindbrain. Scale bar: 3168 µm (D) qPCR analysis of NHE6 and NHE9 in primary murine neurons and astrocytes with mRNA normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent SD determined from triplicate measurements.
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Figure 4: Developmental regulation of NHE9 in mouse and expression in primary brain cells(A) Raw expression levels of NHE9 in various regions of the mouse brain determined from in situ hybridization data obtained from Allen Brain Atlas [Available from: http://mouse.brain-map.org/]. ISOCTX: Isocortex, OLF: Olfactory areas, HPF: Hippocampal formation, CTXsp: Cortical subplate, STR: Striatum, PAL: Pallidum, CB: Cerebellum, TH: Thalamus, HY: Hypothalamus, MB: Midbrain, P: Pons, MY: Medulla. (B) NHE9 gene expression in developing mouse brain characterized by in situ hybridization (ISH) in sagittal plane across four embryonic and three early postnatal ages. Feulgen-HP yellow DNA counterstain, a nuclear stain, was used to add definition to the tissue. This counterstain is used in conjunction with ISH for all data shown except for P56, in order to provide tissue context to the ISH signal which is otherwise difficult to discern due to the very light tissue background for embryonic ISH. Images were obtained from Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org] (C)In situ hybridization data showing expression summary of NHE9 in the various regions of the mouse brain during development, obtained from Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org]. RSP: Rostral secondary prosencephalon, Tel: Telencephalic vesicle, PHy: Peduncular (caudal) hypothalamus, p3: Prosomere 3, p2: Prosomere 2, p1: Prosomere 1, M: Midbrain, PPH: Prepontine hindbrain, PH: Pontine hindbrain, PMH: Pontomedullary hindbrain, MH: Medullary hindbrain. Scale bar: 3168 µm (D) qPCR analysis of NHE6 and NHE9 in primary murine neurons and astrocytes with mRNA normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent SD determined from triplicate measurements.

Mentions: In the wake of the association of NHE9 with autism we sought to investigate the spatiotemporal distribution of NHE9 in the developing and adult mouse brain. Although no single region of the brain has yet been clearly identified as being associated with autism, two decades of magnetic resonance imaging (MRI) studies have implicated cerebellum, frontal cortex, hippocampus and amygdala. Postmortem findings, animal models, and neuroimaging studies further strengthen these observations39,40. In-situ hybridization data of the adult mouse brain obtained from the Allen Brain Atlas41 indicate that the expression levels of NHE9 are highest in the cortex (~27% of total NHE9 expression in the brain), hippocampus (~30%) and the olfactory lobes (~50%) (Figure 4A) compared to the other regions of the brain. Variations of gene expression in the brain may play a crucial role in the behavioral phenotypes observed in autism and suggest a strong association with the cortex, which is the seat of memory, attention, thought, language, and consciousness in the brain. Recent reports comparing autistic and control brains suggested an attenuation of normal differential gene expression between frontal and temporal cortex in autistic brains42. Moreover, as autism is a neurodevelopmental disorder we expect NHE9 to play a functional role during development. Indeed, in situ hybridization data of the developing mouse brain43 revealed differential expression of NHE9 during the various stages of development (Figure 4B). NHE9 expression was consistently high in the prosomere 1(p1) region of the diencephalon in the embryonic forebrain (Figure 4C). In addition to the forebrain, expression levels of NHE9 were also high in the midbrain by postnatal day 4(P4). Although the p1 and midbrain showed high levels of NHE9 expression in P28 pups, highest levels of NHE9 were observed in the telencephalic vesicle of the forebrain. Pontomedullary region of the hindbrain also showed NHE9 expression comparable to the p1 region by P28. Finally, the adult brain is primarily composed of two broad classes of cells: neurons and glial cells. We compared the mRNA transcript levels of NHE9 in neurons and astrocytes, the most abundant macroglial cells in the cortex. NHE9 expression was ~1.2 fold higher in astrocytes relative to the neurons (Figure 4D).


Functional evaluation of autism-associated mutations in NHE9.

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

Developmental regulation of NHE9 in mouse and expression in primary brain cells(A) Raw expression levels of NHE9 in various regions of the mouse brain determined from in situ hybridization data obtained from Allen Brain Atlas [Available from: http://mouse.brain-map.org/]. ISOCTX: Isocortex, OLF: Olfactory areas, HPF: Hippocampal formation, CTXsp: Cortical subplate, STR: Striatum, PAL: Pallidum, CB: Cerebellum, TH: Thalamus, HY: Hypothalamus, MB: Midbrain, P: Pons, MY: Medulla. (B) NHE9 gene expression in developing mouse brain characterized by in situ hybridization (ISH) in sagittal plane across four embryonic and three early postnatal ages. Feulgen-HP yellow DNA counterstain, a nuclear stain, was used to add definition to the tissue. This counterstain is used in conjunction with ISH for all data shown except for P56, in order to provide tissue context to the ISH signal which is otherwise difficult to discern due to the very light tissue background for embryonic ISH. Images were obtained from Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org] (C)In situ hybridization data showing expression summary of NHE9 in the various regions of the mouse brain during development, obtained from Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org]. RSP: Rostral secondary prosencephalon, Tel: Telencephalic vesicle, PHy: Peduncular (caudal) hypothalamus, p3: Prosomere 3, p2: Prosomere 2, p1: Prosomere 1, M: Midbrain, PPH: Prepontine hindbrain, PH: Pontine hindbrain, PMH: Pontomedullary hindbrain, MH: Medullary hindbrain. Scale bar: 3168 µm (D) qPCR analysis of NHE6 and NHE9 in primary murine neurons and astrocytes with mRNA normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent SD determined from triplicate measurements.
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Figure 4: Developmental regulation of NHE9 in mouse and expression in primary brain cells(A) Raw expression levels of NHE9 in various regions of the mouse brain determined from in situ hybridization data obtained from Allen Brain Atlas [Available from: http://mouse.brain-map.org/]. ISOCTX: Isocortex, OLF: Olfactory areas, HPF: Hippocampal formation, CTXsp: Cortical subplate, STR: Striatum, PAL: Pallidum, CB: Cerebellum, TH: Thalamus, HY: Hypothalamus, MB: Midbrain, P: Pons, MY: Medulla. (B) NHE9 gene expression in developing mouse brain characterized by in situ hybridization (ISH) in sagittal plane across four embryonic and three early postnatal ages. Feulgen-HP yellow DNA counterstain, a nuclear stain, was used to add definition to the tissue. This counterstain is used in conjunction with ISH for all data shown except for P56, in order to provide tissue context to the ISH signal which is otherwise difficult to discern due to the very light tissue background for embryonic ISH. Images were obtained from Allen Institute for Brain Science, Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org] (C)In situ hybridization data showing expression summary of NHE9 in the various regions of the mouse brain during development, obtained from Allen Developing Mouse Brain Atlas [Available from: http://developingmouse.brain-map.org]. RSP: Rostral secondary prosencephalon, Tel: Telencephalic vesicle, PHy: Peduncular (caudal) hypothalamus, p3: Prosomere 3, p2: Prosomere 2, p1: Prosomere 1, M: Midbrain, PPH: Prepontine hindbrain, PH: Pontine hindbrain, PMH: Pontomedullary hindbrain, MH: Medullary hindbrain. Scale bar: 3168 µm (D) qPCR analysis of NHE6 and NHE9 in primary murine neurons and astrocytes with mRNA normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent SD determined from triplicate measurements.
Mentions: In the wake of the association of NHE9 with autism we sought to investigate the spatiotemporal distribution of NHE9 in the developing and adult mouse brain. Although no single region of the brain has yet been clearly identified as being associated with autism, two decades of magnetic resonance imaging (MRI) studies have implicated cerebellum, frontal cortex, hippocampus and amygdala. Postmortem findings, animal models, and neuroimaging studies further strengthen these observations39,40. In-situ hybridization data of the adult mouse brain obtained from the Allen Brain Atlas41 indicate that the expression levels of NHE9 are highest in the cortex (~27% of total NHE9 expression in the brain), hippocampus (~30%) and the olfactory lobes (~50%) (Figure 4A) compared to the other regions of the brain. Variations of gene expression in the brain may play a crucial role in the behavioral phenotypes observed in autism and suggest a strong association with the cortex, which is the seat of memory, attention, thought, language, and consciousness in the brain. Recent reports comparing autistic and control brains suggested an attenuation of normal differential gene expression between frontal and temporal cortex in autistic brains42. Moreover, as autism is a neurodevelopmental disorder we expect NHE9 to play a functional role during development. Indeed, in situ hybridization data of the developing mouse brain43 revealed differential expression of NHE9 during the various stages of development (Figure 4B). NHE9 expression was consistently high in the prosomere 1(p1) region of the diencephalon in the embryonic forebrain (Figure 4C). In addition to the forebrain, expression levels of NHE9 were also high in the midbrain by postnatal day 4(P4). Although the p1 and midbrain showed high levels of NHE9 expression in P28 pups, highest levels of NHE9 were observed in the telencephalic vesicle of the forebrain. Pontomedullary region of the hindbrain also showed NHE9 expression comparable to the p1 region by P28. Finally, the adult brain is primarily composed of two broad classes of cells: neurons and glial cells. We compared the mRNA transcript levels of NHE9 in neurons and astrocytes, the most abundant macroglial cells in the cortex. NHE9 expression was ~1.2 fold higher in astrocytes relative to the neurons (Figure 4D).

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