Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia

Abstract

Context: Disturbances in markers of cortical γ-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of γ-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4.

Objective: To compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects.

Design: Real-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex.

Setting: Human brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania.

Participants: Postmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term.

Main outcome measures: Relative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts.

Results: OXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia.

Conclusion: In schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of γ-aminobutyric acid neurotransmission in the prefrontal cortex.

Figure 1

NKCC1 (A), KCC2 (B), OXSR1 (C) and WNK3-1 (D) expression ratio (ER, where ER= 2^−dCTs) values for CR and SZ subjects in the 24-pair (black circles) and 18-pair (white circles) cohorts. Data points below the diagonal unity line represent subject pairs in which the expression level for the gene of interest is greater in the schizophrenia subjects relative to the matched normal comparison subject.

Figure 2

Effect of antipsychotic medication on the transcript expression for OXSR1, WNK3-1 and WNK3-1&2. Expression ratio values (ER) were calculated for each transcript for six triads of monkeys with long-term exposure to placebo (white bars), haloperidol (light grey bars) or olanzapine (dark grey bars).

Figure 3

Mean (bar) and individual (circle) expression ratio (ER) values for OXSR1 (A) and WNK3-1 (B) are shown for the subjects with schizophrenia grouped by potential confounding factors. Neither sex, diagnosis of schizoaffective disorder, suicide, antidepressant medication use at the time of death, use of benzodiazepines or sodium valproate at the time of death, antipsychotic medication use at the time of death, nor diagnosis of substance abuse or dependence at the time of death significantly affected OXSR1 (A) or WNK3-1 (B) transcript expression. Numbers in bars indicate the number of subjects with schizophrenia in each category.

Figure 4

Four most significant Pearson correlations (p<10⁻³) between dCTs for 84 subjects (normal comparison subjects in black circles and subjects with schizophrenia in white circles).

Figure 5

Brightfield photomicrographs showing Nissl staining (panel A) and laminar distribution pattern of OXSR1 immunoreactivity in human DLPFC area 9 for a subject with a short PMI (5.5 hours, panel B) and a long PMI (19.3 hours, panel C). The insert shows a magnification of OXSR1 immunoreactivity in layer 3.

Figure 6

Putative interaction model between NKCC1 and KCC2 and OXSR1 and WNK3-1 kinases. In both panels, the orange bar represents the cell membrane, with the extracellular domain above and the intracellular domain below the bar. The size and orientation of the green arrows indicates the magnitude and direction of Cl⁻ ion flow mediated by NKCC1, KCC2 and GABA-A receptor Cl⁻ channels. (A) In normal adult neurons, intracellular Cl⁻concentration is low due to low levels of NKCC1 and high levels of KCC2. The binding of GABA to GABA-A receptors triggers Cl⁻ entry (Cl⁻ flow is represented by green arrows) and hyperpolarization. (B) In schizophrenia, increased OXSR1 and WNK3-1 kinase levels lead to increased phosphorylation (blue P) and consequently increased NKCC1 activity and decreased KCC2 activity, producing a greater intracellular Cl⁻ concentration. Thus, when GABA-A receptors are activated Cl⁻ influx is reduced (or perhaps reversed) and the nature of GABA neurotransmission is altered.