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Review
. 2014 Jun 23:8:172.
doi: 10.3389/fncel.2014.00172. eCollection 2014.

An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease

Kalyan C Kondapalli  1 , Hari Prasad  1 , Rajini Rao  1
Affiliations
Review

An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease

Kalyan C Kondapalli et al. Front Cell Neurosci. .

Abstract

Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na(+)/H(+) exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder (ADHD), intellectual disability, and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na(+), K(+)) content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo. Drawing upon insights from model organisms and mammalian cells we show how eNHE affect surface expression and function of membrane receptors and neurotransmitter transporters. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

Keywords: ADHD; Christianson syndrome; SLC9A6; SLC9A9; autism; endosomes; sodium proton exchanger; trafficking.

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Figures

Figure 1
Figure 1
The Pump-Leak hypothesis of endosomal pH regulation. Endosomal pH is precisely tuned by a combination of proton pumping or acidification through the V-ATPase and proton leaking or alkalization via endosomal Na+(K+)/H+ exchangers (eNHE), both evolutionarily conserved from yeast to plants and mammals. In mammalian cells, under physiological conditions, the endosomal pH is acidic (≈5.7) relative to the cytoplasmic pH (≈7.2). As per the pump-leak hypothesis, the H+ gradient generated by the V-ATPase (V-type H+-ATPase) drives secondary active transport by eNHE, resulting in cation (Na+ or K+) sequestration coupled to removal of protons from the compartmental lumen. From this model it is intuitive that eNHE activity would provide the range and flexibility to counter fluctuations in subcellular environment. Also, loss-of-function mutations in eNHE would cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways, impacting cargo processing, turnover, and trafficking. In addition to the intricate balance between proton pump and leak pathways, counter-ion (anion and cation) conductances (not shown) also contribute to pH homeostasis. The pH values indicated in the figure were collected from published literature (Casey et al., ; Kondapalli et al., 2013).
Figure 2
Figure 2
Gene Distribution of patient mutations in NHE6 and NHE9. (A,B) The distribution of NHE6 (A) and NHE9 (B) mutations (missense, non-sense, and in-frame deletions) in N-terminal transmembrane domain and the C-terminal regulatory cytoplasmic domain, listed in Table 1, are displayed. The x-axis indicates amino acid locations of NHE6 and NHE9 proteins, while the y-axis displays the number of published literature reports. All mutations in NHE6 are referred to in relation to longer NHE6.1 isoform (NP_001036002.1).
Figure 3
Figure 3
Model-Structures of NHE6 and NHE9 with patient mutations. (A,B) Model of NHE6 (A) and NHE9 (B) are based on methods described in detail elsewhere (Landau et al., ; Kondapalli et al., 2013). Side views of the membrane embedded N-terminal transporter domains are shown, with the cytoplasm below. The TM4–TM11 assembly (colored yellow and green, respectively) is key to the antiport mechanism, and consists of extended segments of the two helices, crossing each other in the middle of the membrane. For clarity, TM3 backbone is shown in stick configuration and a part of the TM1–2 loop of NHE9 was omitted. Positions in which clinical mutations were detected are marked, shown as all-atom spheres and colored according to their ConSurf evolutionary conservation scores (Ashkenazy et al., , http://consurf.tau.ac.il/) as follows: strongly conserved residues (magenta), weakly conserved (white), non-conserved (blue). Many of the conserved sites that show substitutions are situated in highly packed regions, including the vicinity of the TM4–TM11 assembly region. P117 of NHE9, on the other hand, is located in the very long and variable loop connecting TM1 and TM2. A minor substitution to Ile was found in V176 of NHE9, a moderately-conserved hydrophobic residue that is oriented toward the lipid bilayer. Three autism-associated variants in NHE9 (L236S, S438P, V176I; Table 1) have been experimentally determined to be loss of function mutations (Kondapalli et al., 2013), the remainder remain to be screened for changes in function. (C) Sequence alignment of C-terminal tail extensions of human NHE6, human NHE9, and rat NHE9. The positions of human NHE6 and NHE9 disease variants in the C-terminal tail are boxed (Table 1). Missense variants reported in rat models of attention deficit/hyperactivity disorder are also indicated (Zhang-James et al., 2011). Highly conserved RACK1 binding region in the C-terminus of NHE6 and NHE9 and the Angiotensin II receptor (AT2) interacting region in the juxtamembrane portion of the NHE6 C-terminus are displayed.
Figure 4
Figure 4
Expression of eNHE in the hippocampus, cerebral cortex, and cerebellum. (A,B) Brain morphology depicted using cartoon atlas and Nissl staining indicating coronal (A) and sagittal (B) planes of hippocampal formation (HPF), with the functionally distinct CA1, CA2, CA3, and dentate gyrus (DG) subfields. In Situ Hybridization (ISH) images of NHE6 and NHE9 expression in coronal (A) plane and sagittal (B) planes of hippocampus accompanied by false-color heat map adjacent to it, showing enriched eNHE expression in DG and CA1–CA3 (NHE6) and DG and CA1 (NHE9) (A,B, ISH and heat-map). NHE9 expression is also seen in the wall of lateral ventricle (VL) (A, ISH and white arrow in the heat-map). (C) Cartoon atlas and Nissl staining depicting cerebral cortex (CTX) in sagittal plane with labeled cortical layer boundaries. NHE9 is expressed in a gradient in the cortical layer subfield, in contrast to an NHE6 that is strongly expressed throughout the cortex (C, ISH and heat-map). (D) Cartoon atlas and Nissl staining showing cerebellar cortex (CBX) in sagittal plane. Prominent NHE6 expression is seen in the Purkinje cells arranged in a single layer between the molecular (mo) and granular (gr) layers. In contrast, NHE9 shows weak expression in the cerebellum (D, ISH and heat-map). False-color reference heat map (expression mask) scale indicating those cells that have the highest probability of gene expression (from low/blue to high/red). ISH expression data are from Allen Brain Atlas obtained from 56 day (8 weeks) old adult male C57BL/6J mice (available from: http://mouse.brain-map.org/) (Lein et al., 2007).
Figure 5
Figure 5
Expression patterns of eNHE during normal brain development. (A) Heat map of raw expression levels (from low/blue to high/red) of eNHE in various regions of the mouse brain determined from in situ hybridization data obtained from Allen Brain Atlas (http://mouse.brain-map.org/) (Lein et al., 2007). Genetically identical, inbred mice were used to limit individual differences and in situ data were normalized to a specially created reference atlas of mouse brain anatomy to control for differences between brain sections (Lein et al., 2007). Abbreviations: LSC, Lateral septal complex; SDR, Striatum dorsal region; THL, Thalamus; STR, Striatum; SVR, Striatum ventral region; CRB, Cerebellum; OLF, Olfactory bulb; RHP, Retrohippocampal region; PAL, Pallidum; HPF, Hippocampal formation; SAM, Striatum-like amygdalar nuclei; PON, Pons; MID, Midbrain; HTH, Hypothalamus; MED, Medulla; CTX, Cortex; HIP, Hippocampal region. (B) Heat map of average human NHE6 and NHE9 RNA-seq gene expression (from low/blue to high/red) plotted across different brain regions and different development stages which include: prenatal period (embryogenesis to birth), infancy (birth to 1 year), childhood (2–10 years), adolescence (11–20 years), and adulthood (21+ years). NHE6 shows highest expression prenatally during embryonic life, which lessens postnatally and again peaks during adulthood. In contrast, NHE9 expression is far lower embryonically, increases postnatally and peaks in the adulthood. Notably, a strong correlation was observed for NHE6 with NRXN1 a well-known autism candidate gene (red asterisk). (C) Hierarchical clustering and expression heat-map (from low/blue to high/red) of eNHE with 21 genes (Xu et al., 2012) with strong evidence for association with autism showing clustering of eNHE with many synapse associated autism genes during normal brain development. Notably, a strong correlation was observed for NHE6 with NRXN1 a well-known autism candidate gene (red asterisk). RNA-seq gene expression dataset included a total of 578 samples represented as log base 2 of RPKM values across different developmental periods and different brain regions (available from: http://www.brainspan.org) (Sunkin et al., 2013). Abbreviations: BA, Brodmann area; CTX, cortex; pcw, post-conception weeks; mos, postnatal months; yrs, age in years; RPKM, reads per kilobase of exon model per million mapped sequence reads.
Figure 6
Figure 6
Schematic of eNHE function at the synapse. (A) Presynaptic Neuron: eNHE regulate luminal proton content to affect fusion of synaptic vesicles with the presynaptic membrane (Step 1). A role for eNHE in membrane assembly and endocytic retrieval of fused vesicles after the fused membrane undergoes lateral diffusion is depicted (Step 2). eNHE may influence the ratio between electrical and chemical gradients by dissipating ΔpH in favor of ΔΨ, thus controlling neurotransmitter loading (Goh et al., 2011) (Step 3). eNHE may also influence retrograde signaling in response to postsynaptic factors (Step 2; Figure 6B). (B) Postsynaptic Neuron: A model of eNHE function is presented in the context of membrane remodeling at dendritic spines (Step 1). Potential regulation of eNHE by Rab-GAP protein is extrapolated from the yeast ortholog, NHX1 and includes a possible interaction with Las17 ortholog WASP and the Gyp6 ortholog TBC1D5 (Ali et al., ; Kallay et al., 2011). Cytosolic domains of the eNHE bind scaffold protein RACK1, and the NHE6-Rack1 interaction controls receptor recycling in cultured cells (Ohgaki et al., 2008). RAB-mediated fusion and actin reorganization via WASP are required for membrane remodeling. Retrograde signaling occurs via factors secreted by the postsynaptic neuron that bind to receptors on the presynaptic membrane. A role of eNHE in retrograde signaling is shown (Step 2; see also Figure 6A). (C) Astrocyte: eNHE modulate luminal pH of endocytic and exocytic pathways in astrocytes (Kondapalli et al., 2013) to regulate vesicular trafficking, localization and turnover neurotransmitter transporters and neurotropic factor receptors (Step 1). Astrocytes surround synapses at which neurotransmitters are spilled over to stimulate astrocytic receptors leading to propagation of Ca2+ transients in astrocytes. Glial transmitters can be released from astrocytes in a Ca2+ dependent manner and can stimulate extra-synaptic receptors on adjacent neurons, leading to a dynamic modification of synaptic transmission. eNHE may differentially modulate the release of these gliotransmitters from the astrocyte (Step 2). Endosomal pH in astrocytes has been shown to affect cleavage of enzymes essential for Notch signaling, critical for astrocyte migration and differentiation (Valapala et al., 2013). A role for eNHE in these processes essential for migration and differentiation is depicted (Step 3).
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