Altered distribution and localization of organellar Na+/H+ exchangers in postmortem schizophrenia dorsolateral prefrontal cortex

Abstract

Schizophrenia is a complex and multifactorial disorder associated with altered neurotransmission as well as numerous signaling pathway and protein trafficking disruptions. The pH of intracellular organelles involved in protein trafficking is tightly regulated and impacts their functioning. The SLC9A family of Na⁺/H⁺ exchangers (NHEs) plays a fundamental role in cellular and intracellular pH homeostasis. Four organellar NHE isoforms (NHE6-NHE9) are targeted to intracellular organelles involved in protein trafficking. Increased interactions between organellar NHEs and receptor of activated protein C kinase 1 (RACK1) can lead to redistribution of NHEs to the plasma membrane and hyperacidification of target organelles. Given their role in organelle pH regulation, altered expression and/or localization of organellar NHEs could be an underlying cellular mechanism contributing to abnormal intracellular trafficking and disrupted neurotransmitter systems in schizophrenia. We thus characterized organellar NHE expression, co-immunoprecipitation with RACK1, and Triton X-114 (TX-114) phase partitioning in dorsolateral prefrontal cortex of 25 schizophrenia and 25 comparison subjects by Western blot analysis. In schizophrenia after controlling for subject age at time of death, postmortem interval, tissue pH, and sex, there was significantly decreased total expression of NHE8, decreased co-immunoprecipitation of NHE8 (64%) and NHE9 (56%) with RACK1, and increased TX-114 detergent phase partitioning of NHE6 (283%), NHE9 (75%), and RACK1 (367%). Importantly, none of these dependent measures was significantly impacted when comparing those in the schizophrenia group on antipsychotics to those off of antipsychotics for at least 6 weeks at their time of death and none of these same proteins were affected in rats chronically treated with haloperidol. In summary, we characterized organellar NHE expression and distribution in schizophrenia DLPFC and identified abnormalities that could represent a novel mechanism contributing to disruptions in protein trafficking and neurotransmission in schizophrenia.

Fig. 1. Schematic representation of organellar Na⁺/H⁺ exchanger (NHE) distributions and roles in intracellular compartmental pH regulation.

A Organellar NHEs play an integral role in regulating the pH of the organelles involved in protein post-translational modification (PTM) and trafficking. NHE6 and NHE9 are localized to early/recycling and late endosomes, respectively while NHE7 and NHE8 are localized to trans-Golgi network and mid/trans-Golgi stacks, respectively. There is an interplay between NHEs at organelle targets and the plasma membrane (PM) with NHEs being stabilized at the PM by interactions with receptor of activated protein C kinase 1 (RACK1), which is activated by protein kinase C (PKC). Altered expression or distribution of organellar NHEs impacts organelle pH regulation and disrupts protein PTM and trafficking with (B) decreased expression or activity at organelle targets typically leading to acidification, and (C) increased expression or activity at organelle targets typically leading to alkalinization. EE early endosome, LE late endosome, RE recycling endosome, TGN trans-Golgi network.

Fig. 2. Protein expression of NHE6-NHE9 and RACK1 in dorsolateral prefrontal cortex (DLPFC) as measured by Western blot in schizophrenia (SZ) and comparison (COMP) subjects.

A Representative Western blot of NHE6 immunoreactive bands demonstrating three main isoforms: glycosylated oligomers, highly glycosylated monomers, and core glycosylated monomers. B Quantification of each NHE6 isoform normalized to intralane valosin-containing protein (VCP) did not reveal any difference in expression between SZ and COMP. C Representative Western blots of NHE7-NHE9 and RACK1 in SZ and COMP DLPFC. D Quantification of NHE7-NHE9 and RACK1 protein expression normalized to intralane VCP demonstrates a significant decrease in NHE8 expression in SZ DLPFC and no change in NHE7, NHE9, or RACK1 expression between groups. Lines represent group means. *p < 0.05.

Fig. 3. NHE association with RACK1 in schizophrenia dorsolateral prefrontal cortex (DLPFC).

A Representative Western blots for RACK1 and NHE6 as well as RACK1 and NHE7-9 following co-immunoprecipitation (co-IP) with RACK1 antibody in human cortex. Co-IP of RACK1 enriches for RACK1 in IP eluate. NHE6-9 are all present in the RACK1 co-IP eluate suggesting interactions between these transporters and RACK1 in human cortex. No RACK1 enrichment was seen in a control co-IP with pre-immune, non-specific mouse IgG, demonstrating the specificity of co-IP with RACK1 antibody. In addition, RACK1 antibody and IgG alone did not produce immunoreactive bands at the molecular weights (MWs) of our assayed proteins. B Representative Western blots probing for RACK1 and NHE6-NHE9 in SZ and COMP DLPFC. C Quantification of NHE6-NHE9 normalized to intralane RACK1 in RACK1 IP eluate demonstrates decreased association between NHE8/NHE9 and RACK1 in SZ DLPFC with a nonsignificant decrease in NHE7 association with RACK1 between groups (p = 0.06). Lines represent group means. **p < 0.01. IP immunoprecipitation, Super supernatant, IgG immunoglobulin G, COMP comparison, SZ schizophrenia, co-IP co-immunoprecipitation.

Fig. 4. NHE Triton X-114 (TX-114) phase partitioning in schizophrenia dorsolateral prefrontal cortex (DLPFC).

A Representative Western blots demonstrating TX-114 aqueous (AQ) and detergent (DT) phase partitioning of NHE6-NHE9, RACK1, recycling and late endosome markers Rab11 and Rab7, respectively, cytosolic marker GAPDH, and synaptic marker PSD95 in SZ and COMP DLPFC. PSD95 is enriched in the DT fraction while GAPDH, Rab7, and Rab11 are enriched in the AQ fraction. NHE6, NHE9, and RACK1 are present in both AQ and DT fractions but are all present in higher proportion in the AQ fraction. B Quantification of NHE6, NHE9, and RACK1 TX-114 AQ and DT partitioning in SZ and COMP. For the AQ fraction, NHE6, NHE9, and RACK1 were normalized to intralane GAPDH. NHE6, NHE9, and RACK1 were unchanged in the AQ fraction in SZ DLPFC. For the DT fraction, NHE6, NHE9, and RACK1 were normalized to intralane PSD95. NHE6, NHE9, and RACK1 were all increased in the DT fraction in SZ DLPFC. Lines represent group means. *p < 0.05. AQ aqueous phase, DT detergent phase, Syn synaptic marker, Cyto cytosolic marker, LE late endosome marker, RE recycling endosome marker.