Marco B. Rust

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold

K‐Cl co‐transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl− concentration. Kcc3−/− mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3−/− mice displayed reduced seizure threshold and spike‐wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3−/− mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K+ recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

Learning, AMPA receptor mobility and synaptic plasticity depend on n‐cofilin‐mediated actin dynamics

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin‐binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n‐cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long‐term potentiation and long‐term depression. Loss of n‐cofilin‐mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n‐cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1(-/-) mice, decreased in Kcc3(-/-) mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1(-/-) or Kcc3(-/-) mice were of normal density, rbc of Kcc1(-/-)Kcc3(-/-) mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1(-/-)Kcc3(-/-) SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K(+) content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

Neurogenic mechanisms contribute to hypertension in mice with disruption of the K-Cl cotransporter KCC3

The neurodegenerative disorder Andermann syndrome is caused by mutations of the K-Cl cotransporter KCC3. Mice with a targeted disruption of the corresponding gene, Slc12a6, reproduce neurodegeneration of the peripheral and central nervous system (CNS) and display arterial hypertension. Kcc3 is expressed in numerous tissues, including the CNS and vascular smooth muscle cells. As the intracellular chloride concentration may influence myogenic tone and hence blood pressure, we measured the chloride concentration in vascular smooth muscle cells. It was indeed increased in superficial brain arteries and saphenous arteries of Kcc3−/− mice. Isolated saphenous arteries and their third-order branches, however, reacted indistinguishably to changes in intravascular pressure, stimulation of &agr;1-adrenoreceptors, exogenous nitric oxide, or blockade of calcium-activated chloride channels. Likewise, the responses to &agr;1-adrenergic stimulation or exogenous nitric oxide in vivo were identical in both genotypes. These results argue against a major vascular-intrinsic component of arterial hypertension in Kcc3−/− mice. In contrast, either &agr;1-adrenergic blockade or inhibition of ganglionic transmission abolished the difference in arterial blood pressure between both genotypes. This demonstrates a neurogenic component in the maintenance of this phenotype, which is further supported by an increase of urinary norepinephrine and epinephrine excretion in Kcc3−/− mice. Our data indicate that local control of myogenic tone does not require KCC3 and that hypertension in Kcc3−/− mice depends on an elevated sympathetic tone.

ADF/cofilin: a crucial regulator of synapse physiology and behavior.

Actin filaments (F-actin) are the major structural component of excitatory synapses, being present in presynaptic terminals and in postsynaptic dendritic spines. In the last decade, it has been appreciated that actin dynamics, the assembly and disassembly of F-actin, is crucial not only for the structure of excitatory synapses, but also for pre- and postsynaptic physiology. Hence, regulators of actin dynamics take a central role in mediating neurotransmitter release, synaptic plasticity, and ultimately behavior. Actin depolymerizing proteins of the ADF/cofilin family are essential regulators of actin dynamics, and a number of recent studies highlighted their crucial functions in excitatory synapses. In dendritic spines, ADF/cofilin activity is required for spine enlargement during initial long-term potentiation (LTP), but needs to be switched off during spine stabilization and LTP consolidation. Conversely, active ADF/cofilin is needed for spine pruning during long-term depression (LTD). Moreover, ADF/cofilin controls activity-induced synaptic availability of glutamate receptors, and exocytosis of synaptic vesicles. These data show that the activity of ADF/cofilin in synapses needs to be spatially and temporally tightly controlled through several upstream regulatory pathways, which have been identified recently. Hence, ADF/cofilin-controlled actin dynamics emerged as a critical and central regulator of synapse physiology. In this review, I will summarize and discuss our current knowledge on the roles of ADF/cofilin in synapse physiology and behavior, by focusing on excitatory synapses of the mammalian central nervous system.

ADF/Cofilin Controls Synaptic Actin Dynamics and Regulates Synaptic Vesicle Mobilization and Exocytosis

Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.

Attention-Deficit/Hyperactivity Disorder-like Phenotype in a Mouse Model with Impaired Actin Dynamics.

BACKGROUND Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.

Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons

Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell–cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN–BG interaction. Here we demonstrate that the actin‐binding protein profilin1 is essential for CGN–glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.

Chloride cotransporters, chloride homeostasis, and synaptic inhibition in the developing auditory system.

The role of glycine and GABA as inhibitory neurotransmitters in the adult vertebrate nervous system has been well characterized in a variety of model systems, including the auditory, which is particularly well suited for analyzing inhibitory neurotransmission. However, a full understanding of glycinergic and GABAergic transmission requires profound knowledge of how the precise organization of such synapses emerges. Likewise, the role of glycinergic and GABAergic signaling during development, including the dynamic changes in regulation of cytosolic chloride via chloride cotransporters, needs to be thoroughly understood. Recent literature has elucidated the developmental expression of many of the molecular components that comprise the inhibitory synaptic phenotype. An equally important focus of research has revealed the critical role of glycinergic and GABAergic signaling in sculpting different developmental aspects in the auditory system. This review examines the current literature detailing the expression patterns and function (chapter 1), as well as the regulation and pharmacology of chloride cotransporters (chapter 2). Of particular importance is the ontogeny of glycinergic and GABAergic transmission (chapter 3). The review also surveys the recent work on the signaling role of these two major inhibitory neurotransmitters in the developing auditory system (chapter 4) and concludes with an overview of areas for further research (chapter 5).

N-cofilin can compensate for the loss of ADF in excitatory synapses.

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.