Behavioral analysis of Ste20 kinase SPAK knockout mice

Abstract

SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase [16]. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open-field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open-field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission.

Introduction

Sensory afferent fiber neurons, which have their cell bodies located in dorsal root ganglia, abundantly express NKCC1, a Na-K-2Cl cotransporter which functions as a secondary active transport mechanism and accumulates Cl⁻ above equilibrium [1], [41]. Upon GABA release, afferent fiber terminals depolarize, leading to inhibition of incoming action potentials. How this primary afferent depolarization results in synaptic inhibition is still not completely understood. However, loss of NKCC1 results in a redistribution of Cl⁻ and absence of depolarizing GABA responses [41]. As a result, NKCC1 knockout mice exhibit increased response latency to nociceptive stimuli [22], [41]. In agreement with a role for the cotransporters in mediating pain signals, NKCC1 inhibitors have been shown to have anti-nociceptive activity [17], [34]. A recent study demonstrated that peripheral nerve injury (axonotomy) results in increased NKCC1 activity [33]. This enhanced activity was not due to increase NKCC1 expression, but rather due to increased phosphorylation of the cotransporter.

Both NKCC1 and KCC2, a K-Cl cotransporter that actively extrudes Cl⁻ from central neurons, are expressed in spinal cord [26]. Spinal cord dorsal horn neurons maintain relatively high level of KCC2 [24] and, as a consequence, have low [Cl⁻]_i. Postsynaptic inhibition of spinal cord dorsal horn neurons via KCC2 may be critical for controlling the flow of sensory information from the periphery through the spinal cord to the brain. Indeed, several studies have shown that decreased KCC2 expression in the spinal cord is associated with increased pain perception [3], [20], [23], [25], [28], [47].

SPAK (Ste20-related Proline Alanine rich Kinase) and OSR1 (Oxidative-Stress Response 1) are mammalian protein kinases involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both the central and peripheral nervous systems. In previous work, we identified SPAK and OSR1 as regulators of the Na-K-2Cl cotransporters NKCC1 [13], [14], [32]. The kinases share high homology in both their catalytic and regulatory domains. Although they belong to a large group of mammalian kinases related to the yeast Ste20p protein, their regulatory domain is unique, indicating a unique function [6], [9]. We have recently examined the expression of the two Ste20 kinases in dorsal root ganglion neurons and found that both kinases are expressed in similar amounts and that each kinase participates equally in the regulation of the cotransporter [16]. As SPAK knockout mice are viable, we were able to analyze NKCC1 activity in the absence of SPAK function and observed that the cotransporter function was reduced by half. A similar study of OSR1 function in native DRG neurons was not possible as the OSR1 knockout mouse is embryonic lethal. SPAK is also expressed in gray and white matter of the spinal cord and brain [31]. SPAK not only controls NKCC1 function, but also affects KCC2 function in a reciprocal fashion [14]. The regulatory and expression pattern of SPAK suggests that it influences intracellular Cl⁻ concentration and thus the amplitude/direction of the GABA response. The present study is the first to characterize the impact of SPAK deletion on general behavioral function.