KCC2 regulates actin dynamics in dendritic spines via interaction with ß-PIX.

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor ß-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.

Distribution of neuronal KCC2a and KCC2b isoforms in mouse CNS.

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters in mature central nervous system (CNS). The KCC2 gene produces two isoforms, KCC2a and KCC2b, that differ in their N-termini. Increase of KCC2b in the cortex underlies the developmental shift in γ-aminobutyric acid (GABA)ergic responses, whereas the physiological role of KCC2a is still poorly characterized. The two KCC2 isoforms show equal distribution in mouse brainstem neurons at birth; however their postnatal expression patterns, and the subcellular localization of KCC2a, have not yet been described. Here, we compared the pattern of KCC2a and KCC2b expression in different regions of postnatal mouse CNS by immunohistochemistry by using isoform-specific antibodies. Tissue from KCC2a isoform-specific knockout mice was used as a negative control. KCC2b expression increased postnatally and was widely expressed in adult brain. KCC2a immunoreactivity was low or absent in most parts of the adult cortex, hippocampus, thalamus, and cerebellar cortex. Both isoforms were widely present in the developing and mature hypothalamus, a large part of the brainstem, and the spinal cord. A notable exception was the lack of KCC2a staining in the brainstem auditory system. At the subcellular level, the isoforms were only partially colocalized. In neuronal somas, KCC2b immunoreactivity was concentrated at the plasma membrane, whereas KCC2a signal was not. Moreover, although both isoforms were expressed in microtubule-associated protein (MAP)2-positive dendrites, they appeared in non-overlapping dendritic compartments. The results, together with those of previous studies, suggest that KCC2a and KCC2b have overlapping roles in neonatal neurons but presumably different roles in mature neurons.

Interactions between ICAM-5 and ß1 integrins regulate neuronal synapse formation.

Intercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems. ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remains poorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synaptic contacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies against ICAM-5 and ß1 integrins altered spine maturation. Furthermore, we found that ß1 integrins serve as binding partners for ICAM-5. ß1 integrins were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. ß1 integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, ß1 integrins covered the mushroom spines. Loss of ß1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage decreased or increased when the interaction between ICAM-5 and ß1 integrins was potentiated or weakened, respectively, using antibodies. These results suggest that the interaction between ICAM-5 and ß1 integrins is important in formation of functional synapses.

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis.

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.