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Leptin-induced dynamic alterations in the actin cytoskeleton mediate the activation and synaptic clustering of BK channels

Neuroscience Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK

¹ Correspondence: E-mail: j.z.harvey{at}dundee.ac.uk

SPECIFIC AIMS

Because previous studies have shown that the activity of both peripheral and neuronal BK channels can be regulated by actin dynamics, we examined the role of the actin cytoskeleton in the activation of hippocampal BK channels by the hormone leptin.

PI 3-kinase links leptin receptor activation to stimulation of BK channels and subsequent inhibition of hippocampal hyperexcitability. To determine the downstream targets of PI 3-kinase and whether leptin receptor driven changes in the levels of PtdIns(3,4,5)P₃ drive the alterations in actin dynamics, we assessed the effects of leptin on the subcellular distribution of PtdIns(3,4,5)P₃ and BK channels in hippocampal neurons.

PRINCIPAL FINDINGS

1. PI 3-kinase-driven disassembly of actin filaments underlies activation of BK channels by leptin
Using cell-attached recording techniques, we examined the effects of stabilizing actin filaments with jasplakinolide on the ability of leptin to activate BK channels in hippocampal neurons. In neurons exposed to jasplakinolide (10 nM; 15 min), application of leptin (10 nM; via the patch pipette) failed to alter BK channel activity such that the mean channel activity (NfPo) 2–4 min after obtaining the cell-attached configuration (0.07±0.02) did not differ significantly from that obtained after leptin treatment (0.069±0.025; n=5; P>0.05). This contrasts with the effects of leptin in the absence of jasplakinolide as leptin stimulated an increase in BK channel activity from 0.06 ± 0.03 (at 2–4 min) to 0.17 ± 0.10 (at 15–17 min; n=8; P<0.01). Application of specific actin-disrupting agents (latrunculin B or cytochalasin D), but not antimicrotubule agents (nocodazole), increased the activity of BK channels in these neurons. Thus, these data indicate that BK channel activation by leptin is likely to involve disruption of actin filaments.

One consequence of BK channel activation by leptin is the inhibition of hyperexcitability in a hippocampal culture model. Thus, we determined whether stabilization of actin filaments prevented the functional effects of leptin on hyperexcitability induced in hippocampal cultures after perfusion of Mg²⁺-free medium. The ability of leptin to inhibit the mean increase in Ca²⁺ levels induced by removal of Mg²⁺ (to 58.3±5.3%; n=221) was completely attenuated by jasplakinolide (to 1.93±1.25%; n=91; P>0.001), suggesting that leptin inhibition of hyperexcitability involved actin disruption. In support of this, disruption of actin filaments with either cytochalasin D (50 µM) or latrunculin B (10 µM) mimicked the actions of leptin resulting in comparable reductions in the enhanced levels of Ca²⁺.

Thus, BK channel activation by the hormone leptin is dependent on the actin cytoskeleton, as it is prevented by actin filament stabilization and mimicked by actin disruption. Moreover, the ability of leptin to inhibit hippocampal hyperexcitability is dependent on the actin cytoskeleton.

2. Leptin promotes rearrangement of actin filaments via a PI 3-kinase-driven pathway
To demonstrate directly that leptin can reorganize actin filaments, alexa 488-conjugated phalloidin (alexa-phalloidin), a fluorescently labeled marker of polymerized actin filaments, was used to visualize the effects of leptin on actin dynamics. Application of leptin (50 nM; 5 min) altered the pattern of alexa-phalloidin labeling such that it became more punctate with less diffuse staining but had little effect on the intensity of staining (n=10). In contrast, exposure to leptin for 30 min induced a marked reduction in the intensity of alexa-phalloidin staining at somata (to 79±3.0% of control; n=12; P<0.05) and processes (to 65±2.1% of control; n=12; P<0.05). In a manner similar to leptin, latrunculin B (n=10) and cytochalasin D (n=10) also altered the pattern of alexa-phalloidin staining, such that it became more punctate. However, the actin disrupters had no effect on the overall intensity of alexa-phalloidin labeling.

Since PI 3-kinase underlies leptin activation of hippocampal BK channels, the role of this enzyme in leptin’s action on actin dynamics was assessed. In neurons treated with LY294002 (10 µM) or wortmannin (50 nM), application of leptin (50 nM; 30 min) failed to reduce the intensity of alexa-phalloidin staining in the processes and somata. The leptin-induced increase in clustering of actin filaments was also driven by a PI 3-kinase-dependent process as exposure to either wortmannin (n=9) or LY294002 (n=11) prevented these effects. In contrast, the Ras-Raf-MAPK signaling cascade, another potential downstream target of neuronal leptin receptors, is unlikely to contribute to the observed effects of leptin as PD98059 (10 µM), a specific inhibitor of MAPK activation, failed to influence the ability of leptin to increase actin filament clustering (n=12) and reduce alexa-phalloidin staining (n=10). Thus, these data indicate that leptin destabilizes actin filaments via a PI 3-kinase-dependent process.

3. Leptin increases PtdIns(3,4,5)P₃ levels at synapses and promotes clustering of BK channels at synapses
Because PtdIns(3,4,5)P₃ is the main product of PI 3-kinase activity, we examined the effects of leptin on PtdIns(3,4,5)P₃ immunolabeling in hippocampal neurons. In control conditions, PtdIns(3,4,5)P₃ labeling was uniformly distributed throughout the plasma membrane, somata, and processes of hippocampal neurons. Exposure to leptin (50 nM; 5 min) resulted in a significant increase in PtdIns(3,4,5)P₃ staining throughout the cell such that the mean intensity of staining at the plasma membrane, cytosol, and processes was 150 ± 7.0% (n=10; P<0.05), 155 ± 9.0% (n=10; P<0.01), and 126 ± 1.9% (n=10; P<0.05), respectively. After exposure to leptin, sites of strong, localized PtdIns(3,4,5)P₃ labeling were associated with synapses. Longer exposures to leptin (50 nM; 30 min) increased PtdIns(3,4,5)P₃ levels such that plasma membrane levels increased to 140 ± 13% of control (n=16; P<0.05), whereas levels increased to 169 ± 19% of control (n=16; P<0.05) in processes. The leptin-induced increases in PtdIns(3,4,5)P₃ levels were driven by PI 3-kinase activation as prior treatment with either LY294002 (10 µM; n=12) or wortmannin (50 nM; n=12) blocked the effects of leptin on PtdIns(3,4,5)P₃ staining.

We have shown that a PI 3-kinase-driven pathway underlies the leptin-induced reorganization of actin filaments and increase in PtdIns(3,4,5)P₃ staining, but it is not clear whether the elevations in PtdIns(3,4,5)P₃ are responsible for the alterations in actin dynamics. Dual-labeling approaches were used to simultaneously compare the effects of leptin on both parameters. In control conditions, leptin (50 nM; 30 min) induced a significant decrease in alexa-phalloidin staining, an effect that was accompanied by a correlated increase in PtdIns(3,4,5)P₃ staining (n=15), suggesting that the increase in PtdIns(3,4,5)P₃ staining is accompanied by a parallel shift toward actin disassembly. To determine whether the increase in PtdIns(3,4,5)P₃ staining occurs prior to actin destabilization, we compared the effects of leptin in the presence of jasplakinolide to prevent actin depolymerization. In neurons exposed to jasplakinolide, the ability of leptin to promote actin depolymerization but not increase PtdIns(3,4,5)P₃ levels was markedly attenuated (n=15), suggesting that the changes in PtdIns(3,4,5)P₃ levels occur prior to the alterations in actin dynamics.

Since the BK channel subunit is the cellular target for leptin, we examined whether the increase in PtdIns(3,4,5)P₃ levels induced by leptin occurred in close proximity to BK subunits. In the absence of leptin, BK staining was evident throughout the neuron with granules of labeling on fine neurites. A small proportion of these sites were associated with hot spots of PtdIns(3,4,5)P₃ (21.0±3.2% colocalization; n=34) or synapsin-1 (44.3±3.0% colocalization; n=57) immunolabeling. After exposure to leptin, BK staining was more punctate, and this was associated with an increase in the degree of colocalization with PtdIns(3,4,5)P₃ (59.1±4.1%; n=41; P<0.01) or synapsin-1 (72.5±2.6%; n=57; P<0.001), suggesting that leptin increases the density of BK channels at hippocampal synapses. The ability of leptin to cluster BK channels at synapsin-1 positive sites was driven by dynamic changes in the actin cytoskeleton as latrunculin B mimicked the effects of leptin (69.2±1.6% colocalization; n=15; P<0.01), whereas jasplakinolide completely prevented the effects of leptin (49.7±1.7% colocalization; n=21; P>0.05).

CONCLUSIONS AND SIGNIFICANCE

Here we show that BK channel activation by the hormone leptin is dependent on the actin cytoskeleton as it is prevented by stabilizing actin filaments and mimicked by actin disruption. Fluorescent labeling of polymerized actin filaments revealed that leptin promotes rapid rearrangement of actin filaments via a PI 3-kinase-driven signaling cascade, an action paralleled by discrete increases in PtdIns(3,4,5)P₃ levels in close proximity to BK channels. After exposure to leptin, there was also an actin-dependent increase in the degree of BK channel immunolabeling that colocalized with synaptic markers. These data are consistent with the notion that leptin activates BK channels in the hippocampus via PI 3-kinase-driven reorganization of actin filaments and subsequent clustering of BK channels at synapses.

In this study we provide evidence that leptin induced an increase in BK staining that colocalized with the synaptic marker synapsin-1. Thus, it is likely that an increase in the density of functional synaptic BK channels contributes to the enhanced channel activity induced by leptin. It is well established that BK channels play an important role in regulating neuronal excitability. In the hippocampus, BK channels are located at presynaptic terminals and somatodendritic regions, and their role is influenced by their subcellular localization. Leptin, via activation of BK channels, potently inhibits a Mg²⁺-free model of hyperexcitability in the hippocampus. As the aberrant synaptic activity that develops following reductions in extracellular Mg²⁺ can limit the viability of neurons and lead to cell death, leptin via promoting actin destabilization may be a potential neuroprotective agent. In support of this, actin disruption protects hippocampal neurons from glutamate-induced neurotoxicity. Gelsolin, an actin severing agent, has potential anti-apoptotic actions as both glutamate toxicity and seizure-induced damage to hippocampal neurons are exacerbated in gelsolin-deficient mice. Thus, these data indicate that leptin activates BK channels in the hippocampus via a complex cascade of signaling events that culminate in rapid changes in actin dynamics and the cellular localization of BK channels. As leptin is capable of inhibiting hippocampal hyperexcitability via activation of BK channels, these findings have important implications for the role of this hormone in reducing unregulated excitability in the brain.

View larger version (19K): [in this window] [in a new window]   Figure 1. Schematic representation of the complex signaling pathways linking leptin receptor activation to BK channel activation. After leptin binding to the leptin receptor (Ob-Rb), PI 3-kinase is activated, which in turn promotes the phosphorylation of PtdIns(4,5,)P2 into PtdIns(3,4,5)P3. The leptin-induced rise in the levels of PtdIns(3,4,5)P3 result in the depolymerization of filamentous actin into globular actin and the subsequent activation and clustering of BK channels at synapses.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4166fje;

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