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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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Subcellular Localization and Functional Analysis of NHE9(A) qPCR analysis of NHE6 and NHE9 mRNA in primary cortical astrocytes, normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent standard deviation determined from triplicate measurements. Baseline expression of NHE9 is significantly lower than NHE6 (note that the 8-fold difference corresponds to 3 cycles of PCR amplification on Log2 scale). (B) qPCR analysis showing the efficacy of overexpression of (NHE9) and shRNA knock-down (NHE9 and NHE6) in primary astrocyte culture. The data are plotted as average fold-change of mRNA levels relative to control levels, with standard deviations determined from triplicate measurements. (C) Subcellular localization of NHE9 in primary cultured cortical astrocytes determined by immunofluorescence confocal microscopy (63x objective) after fixation with 4% PFA. Top, NHE9-GFP (green) partly localizes with early endosome marker, EEA1 (red) as seen in the Merge. Middle, NHE9-GFP (green) partly localizes with recycling endosome marker, Rab11 (red) as seen in the Merge. Bottom, NHE9-GFP (green) does not localize with late endosome marker, LBPA (red). (D) Orthogonal views of subcellular localization of NHE9 from merged images in (C). (E) Overlapping subcellular localization of NHE6-GFP (green) and NHE9-DsRed (red) in primary cultured cortical astrocytes, as seen in Merge and (F) Orthogonal view. Scale bars for C and E: 50 µm
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Figure 5: Subcellular Localization and Functional Analysis of NHE9(A) qPCR analysis of NHE6 and NHE9 mRNA in primary cortical astrocytes, normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent standard deviation determined from triplicate measurements. Baseline expression of NHE9 is significantly lower than NHE6 (note that the 8-fold difference corresponds to 3 cycles of PCR amplification on Log2 scale). (B) qPCR analysis showing the efficacy of overexpression of (NHE9) and shRNA knock-down (NHE9 and NHE6) in primary astrocyte culture. The data are plotted as average fold-change of mRNA levels relative to control levels, with standard deviations determined from triplicate measurements. (C) Subcellular localization of NHE9 in primary cultured cortical astrocytes determined by immunofluorescence confocal microscopy (63x objective) after fixation with 4% PFA. Top, NHE9-GFP (green) partly localizes with early endosome marker, EEA1 (red) as seen in the Merge. Middle, NHE9-GFP (green) partly localizes with recycling endosome marker, Rab11 (red) as seen in the Merge. Bottom, NHE9-GFP (green) does not localize with late endosome marker, LBPA (red). (D) Orthogonal views of subcellular localization of NHE9 from merged images in (C). (E) Overlapping subcellular localization of NHE6-GFP (green) and NHE9-DsRed (red) in primary cultured cortical astrocytes, as seen in Merge and (F) Orthogonal view. Scale bars for C and E: 50 µm

Mentions: Astrocytes are critical for the long-term modulation of neuronal synapses as well as acute clearance of the excitatory neurotransmitter, glutamate, from the synaptic cleft34. In animal models of autism, astrocytic clearance of glutamate is altered and glutamate transporter levels decreased44. Elevated levels of glutamate in the synapse trigger seizures, and seizures are well known to be co-morbid with autism. As shown by Morrow et al.29, a significant subset of NHE9 variants was associated with both autism and seizures. Furthermore, elevated brain glutamate levels are observed in patients with mutations in the closely related ortholog NHE619. Therefore, to confirm our findings in yeast and extend the analysis of autism-associated variants in NHE9 to a neurobiological model we chose to evaluate function in astrocytes19. We began by evaluating expression levels of NHE9 and NHE6 isoforms in primary mouse astrocytes. Transcript analysis revealed the presence of both NHE6 and NHE9 in cDNA extracts from astrocytes (Figure 5A) as well as in neurons (Figure 4D). Knockdown of NHE9 (by ~80%) in the astrocytes did not alter transcript levels for NHE6, although a modest compensatory increase in NHE9 levels (15%, p= 0.004; Student's t-test, n= three biological replicates) was consistently observed upon knockdown of NHE6 (Figure 5B). We also engineered lentiviral-mediated overexpression of NHE9-GFP (Figure 5B). NHE9-GFP co-localized in part with markers for the early endosome EEA1 (fractional colocalization, 0.11 ± 0.06 SD, n=46) and more extensively with the recycling endosome marker Rab11 (0.46 ± 0.25 SD, n=71) by immunofluorescence (Figure 5C–D, top and middle panel). No NHE9-GFP was observed in the late endosome, as evidenced by lack of co-localization with lysobisphosphatidic acid (−0.01 ± 0.02 SD, n=50; Figure 5C–D, bottom panel).


Functional evaluation of autism-associated mutations in NHE9.

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

Subcellular Localization and Functional Analysis of NHE9(A) qPCR analysis of NHE6 and NHE9 mRNA in primary cortical astrocytes, normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent standard deviation determined from triplicate measurements. Baseline expression of NHE9 is significantly lower than NHE6 (note that the 8-fold difference corresponds to 3 cycles of PCR amplification on Log2 scale). (B) qPCR analysis showing the efficacy of overexpression of (NHE9) and shRNA knock-down (NHE9 and NHE6) in primary astrocyte culture. The data are plotted as average fold-change of mRNA levels relative to control levels, with standard deviations determined from triplicate measurements. (C) Subcellular localization of NHE9 in primary cultured cortical astrocytes determined by immunofluorescence confocal microscopy (63x objective) after fixation with 4% PFA. Top, NHE9-GFP (green) partly localizes with early endosome marker, EEA1 (red) as seen in the Merge. Middle, NHE9-GFP (green) partly localizes with recycling endosome marker, Rab11 (red) as seen in the Merge. Bottom, NHE9-GFP (green) does not localize with late endosome marker, LBPA (red). (D) Orthogonal views of subcellular localization of NHE9 from merged images in (C). (E) Overlapping subcellular localization of NHE6-GFP (green) and NHE9-DsRed (red) in primary cultured cortical astrocytes, as seen in Merge and (F) Orthogonal view. Scale bars for C and E: 50 µm
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Figure 5: Subcellular Localization and Functional Analysis of NHE9(A) qPCR analysis of NHE6 and NHE9 mRNA in primary cortical astrocytes, normalized to two reference genes (GAPDH and 18S RNA) and expressed relative to NHE9 mRNA level. Error bars represent standard deviation determined from triplicate measurements. Baseline expression of NHE9 is significantly lower than NHE6 (note that the 8-fold difference corresponds to 3 cycles of PCR amplification on Log2 scale). (B) qPCR analysis showing the efficacy of overexpression of (NHE9) and shRNA knock-down (NHE9 and NHE6) in primary astrocyte culture. The data are plotted as average fold-change of mRNA levels relative to control levels, with standard deviations determined from triplicate measurements. (C) Subcellular localization of NHE9 in primary cultured cortical astrocytes determined by immunofluorescence confocal microscopy (63x objective) after fixation with 4% PFA. Top, NHE9-GFP (green) partly localizes with early endosome marker, EEA1 (red) as seen in the Merge. Middle, NHE9-GFP (green) partly localizes with recycling endosome marker, Rab11 (red) as seen in the Merge. Bottom, NHE9-GFP (green) does not localize with late endosome marker, LBPA (red). (D) Orthogonal views of subcellular localization of NHE9 from merged images in (C). (E) Overlapping subcellular localization of NHE6-GFP (green) and NHE9-DsRed (red) in primary cultured cortical astrocytes, as seen in Merge and (F) Orthogonal view. Scale bars for C and E: 50 µm
Mentions: Astrocytes are critical for the long-term modulation of neuronal synapses as well as acute clearance of the excitatory neurotransmitter, glutamate, from the synaptic cleft34. In animal models of autism, astrocytic clearance of glutamate is altered and glutamate transporter levels decreased44. Elevated levels of glutamate in the synapse trigger seizures, and seizures are well known to be co-morbid with autism. As shown by Morrow et al.29, a significant subset of NHE9 variants was associated with both autism and seizures. Furthermore, elevated brain glutamate levels are observed in patients with mutations in the closely related ortholog NHE619. Therefore, to confirm our findings in yeast and extend the analysis of autism-associated variants in NHE9 to a neurobiological model we chose to evaluate function in astrocytes19. We began by evaluating expression levels of NHE9 and NHE6 isoforms in primary mouse astrocytes. Transcript analysis revealed the presence of both NHE6 and NHE9 in cDNA extracts from astrocytes (Figure 5A) as well as in neurons (Figure 4D). Knockdown of NHE9 (by ~80%) in the astrocytes did not alter transcript levels for NHE6, although a modest compensatory increase in NHE9 levels (15%, p= 0.004; Student's t-test, n= three biological replicates) was consistently observed upon knockdown of NHE6 (Figure 5B). We also engineered lentiviral-mediated overexpression of NHE9-GFP (Figure 5B). NHE9-GFP co-localized in part with markers for the early endosome EEA1 (fractional colocalization, 0.11 ± 0.06 SD, n=46) and more extensively with the recycling endosome marker Rab11 (0.46 ± 0.25 SD, n=71) by immunofluorescence (Figure 5C–D, top and middle panel). No NHE9-GFP was observed in the late endosome, as evidenced by lack of co-localization with lysobisphosphatidic acid (−0.01 ± 0.02 SD, n=50; Figure 5C–D, bottom panel).

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