Therapeutic effects of KCC2 chloride transporter activation on detrusor overactivity in mice with spinal cord injury.

This study aimed to clarify whether downregulation of K+-Cl- cotransporter 2 (KCC2) in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). SCI was produced by Th8-9 spinal cord transection in female C57BL/6 mice. At 4 wk after SCI, CLP290, a KCC2 activator, was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC2 expression in cholinergic preganglionic parasympathetic neurons in the SPN was examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in the spinal cord slice preparations. DO evident as nonvoiding contractions was significantly reduced by CLP290 treatment in SCI mice. The number of c-fos-positive cells and coexpression of c-fos in choline acetyltransferase-positive cells were decreased in the SPN region of the SCI CLP290-treated group versus the SCI vehicle-treated group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons and normalized fluorescence intensity of KCC2 in choline acetyltransferase-positive SPN neurons was decreased in the SCI vehicle-treated group versus the spinal intact vehicle-treated group but recovered in the SCI CLP290-treated group. Extracellular recordings showed that CLP290 suppressed the high-frequency firing activity of SPN neurons in SCI mice. These results indicated that SCI-induced DO is associated with downregulation of KCC2 in preganglionic parasympathetic neurons and that activation of KCC2 transporters can reduce DO, increase KCC2 expression in preganglionic parasympathetic neurons, and decrease neuronal firing of SPN neurons in SCI mice.NEW & NOTEWORTHY This study is the first report to suggest that activation of the Cl- transporter K+-Cl- cotransporter 2 may be a therapeutic modality for the treatment of spinal cord injury-induced detrusor overactivity by targeting bladder efferent pathways.

De novo variants in CAMK2A and CAMK2B cause neurodevelopmental disorders.

Objective: α (CAMK2A) and ß (CAMK2B) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α- and ß-CaMKII variants in neurodevelopmental disorders. Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis. Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α. By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K+ currents, which facilitated spike repolarization of single action potentials. Interpretation: Our data highlight the importance of CaMKII α and CaMKII ß and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K+ currents as a possible pathophysiological basis.

Development and regulation of chloride homeostasis in the central nervous system.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the mature central nervous system (CNS). The developmental switch of GABAergic transmission from excitation to inhibition is induced by changes in Cl(-) gradients, which are generated by cation-Cl(-) co-transporters. An accumulation of Cl(-) by the Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) increases the intracellular Cl(-) concentration ([Cl(-)]i) such that GABA depolarizes neuronal precursors and immature neurons. The subsequent ontogenetic switch, i.e., upregulation of the Cl(-)-extruder KCC2, which is a neuron-specific K(+)-Cl(-) co-transporter, with or without downregulation of NKCC1, results in low [Cl(-)]i levels and the hyperpolarizing action of GABA in mature neurons. Development of Cl(-) homeostasis depends on developmental changes in NKCC1 and KCC2 expression. Generally, developmental shifts (decreases) in [Cl(-)]i parallel the maturation of the nervous system, e.g., early in the spinal cord, hypothalamus and thalamus, followed by the limbic system, and last in the neocortex. There are several regulators of KCC2 and/or NKCC1 expression, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF), and cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, regionally different expression of these regulators may also contribute to the regional developmental shifts of Cl(-) homeostasis. KCC2 and NKCC1 functions are also regulated by phosphorylation by enzymes such as PKC, Src-family tyrosine kinases, and WNK1-4 and their downstream effectors STE20/SPS1-related proline/alanine-rich kinase (SPAK)-oxidative stress responsive kinase-1 (OSR1). In addition, activation of these kinases is modulated by humoral factors such as estrogen and taurine. Because these transporters use the electrochemical driving force of Na(+) and K(+) ions, topographical interaction with the Na(+)-K(+) ATPase and its modulators such as creatine kinase (CK) should modulate functions of Cl(-) transporters. Therefore, regional developmental regulation of these regulators and modulators of Cl(-) transporters may also play a pivotal role in the development of Cl(-) homeostasis.

Accumulation of GABAergic neurons, causing a focal ambient GABA gradient, and downregulation of KCC2 are induced during microgyrus formation in a mouse model of polymicrogyria.

Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase-green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl(-) transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl(-) concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca(2+) oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca(2+) oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.

A perturbation of multimodal GABA functions underlying the formation of focal cortical malformations: assessments by using animal models.

In order to study how the formation of focal cortical malformations is attributed to perturbation of developmentally multimodal γ-aminobutyric acid (GABA) functions, we made a focal cortical freeze-lesion on a rodent cerebral cortex at P0 (postnatal day 0). The microgyrus was formed at P7. GABA neurons were accumulated in the region surrounding necrosis at P4. Cortical plate cells born at E17.5 gathered, surrounding the GABA neurons, forming the cell dense portions in layer 2 of the microgyrus. Ambient GABA level was increased in the area corresponding to populated GABA neurons at P4. A KCC2 expression was downregulated, whereas an NKCC1 expression was upregulated in both the gathering GABA and cortical plate neurons, suggesting these cells had high intracellular Cl(-) concentration rendering GABA action depolarizing. GABAA receptor activation was involved in Ca(2+) oscillation in these gathering cells. In vivo blockade of GABAA receptor prevented the above characteristic pattern of cell accumulation and hence microgyrus formation. Thus, neonatal freeze-lesion causes characteristic accumulation of differential populations of neurons preceded by characteristic release of GABA at an early stage, which induces GABAA receptor-mediated depolarization and Ca(2+) oscillation. This paracrine/autocrine GABA may underlie the formation of neocortical malformations such as polymicrogyria.

Phosphodiesterase 4 inhibition enhances the dopamine D1 receptor/PKA/DARPP-32 signaling cascade in frontal cortex.

RATIONALE: Alteration of dopamine neurotransmission in the prefrontal cortex, especially hypofunction of dopamine D1 receptors, contributes to psychotic symptoms and cognitive deficit in schizophrenia. D1 receptors signal through the cAMP/PKA second messenger cascade, which is modulated by phosphodiesterase (PDE) enzymes that hydrolyze and inactivate cyclic nucleotides. Though several PDEs are expressed in cortical neurons, the PDE4 enzyme family (PDE4A-D) has been implicated in the control of cognitive function. The best studied isoform, PDE4B, interacts with a schizophrenia susceptibility factor, disrupted in schizophrenia 1 (DISC1). OBJECTIVES: We explore the control of mouse frontal cortex dopamine D1 receptor signaling and associated behavior by PDE4. RESULTS: Inhibition of PDE4 by rolipram induced activation of cAMP/PKA signaling in cortical slices and in vivo, leading to the phosphorylation of DARPP-32 and other postsynaptic and presynaptic PKA-substrates. Rolipram also enhanced DARPP-32 phosphorylation invoked by D1 receptor activation. Immunohistochemical studies demonstrated PDE4A, PDE4B, and PDE4D expression in DARPP-32-positive neurons in layer VI of frontal cortex, most likely in D1 receptor-positive, glutamatergic corticothalamic pyramidal neurons. Furthermore, the ability of rolipram treatment to improve the performance of mice in a sensorimotor gating test was DARPP-32-dependent. CONCLUSIONS: PDE4, which is co-expressed with DARPP-32 in D1 receptor-positive cortical pyramidal neurons in layer VI, modulates the level of D1 receptor signaling and DARPP-32 phosphorylation in the frontal cortex, likely influencing cognitive function. These biochemical and behavioral actions of PDE4 inhibitors may contribute to the hypothesized antipsychotic actions of this class of compounds.

KCC2 was downregulated in small neurons localized in epileptogenic human focal cortical dysplasia.

Focal cortical dysplasia (FCD), which is characterized histologically by disorganized cortical lamination and large abnormal cells, is one of the major causes of intractable epilepsies. γ-aminobutyric acid (GABA)(A) receptor-mediated synchronous depolarizing potentials have been observed in FCD tissue. Since alterations in Cl(-) homeostasis might underlie these depolarizing actions of GABA, cation-Cl(-) cotransporters could play critical roles in the generation of these abnormal actions. We examined the expression patterns of NKCC1 and KCC2 by in situ hybridization histochemistry and immunohistochemistry in FCD tissue obtained by surgery from patients with intractable epilepsy. KCC2 mRNA and protein were expressed not only in non-dysplastic neurons in histologically normal portions located in the periphery of the excised cortex, but also in dysplastic cells in FCD tissue. The levels of KCC2 mRNA and protein were significantly decreased in the neurons around large abnormal neurons (giant neurons), but not in giant neurons, compared with non-dysplastic neurons. The neurons localized only around giant neurons significantly smaller than non-dysplastic neurons. However NKCC1 expression did not differ among these cell types. These results suggest that the intracellular Cl(-) concentration ([Cl(-)](i)) of small neurons might increase, so that depolarizing GABA actions could occur in the FCD tissue of epileptic foci.

Induction of amyloid beta accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells.

PURPOSE: To investigate the intracellular mechanisms that induce amyloid beta (Abeta) accumulation and angiogenesis in the human retinal pigment epithelial cell line ARPE19. METHODS: The authors used two endoplasmic reticulum (ER) stress-inducing reagents, thapsigargin (TG), which inhibits the sarcoplasmic/endoplasmic calcium (Ca)2+-ATPase, and tunicamycin (TM), which inhibits N-linked glycosylation. The expression pattern of Abeta-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of Abeta metabolism-related factors and angiogenic factors were evaluated by real-time RT-PCR and Western blot (VEGF). Expression of caspase-4 was examined by real-time RT-PCR and Western blot to evaluate the effect of the ER stressor. Intracellular Ca elevation by TG was evaluated by Ca2+ imaging experiments. Dimethyl sulfoxide and staurosporine were used as a nonreagent control and as an apoptosis-inducing reagent through mitochondria not ER, respectively. RESULTS: TG-treated ARPE19 cells increased the mRNA expression of Abeta production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for Abeta. TG-treated ARPE19 cells produced increases in VEGF, TNF-alpha, TACE mRNA, and VEGF protein expressions and a decrease in PEDF mRNA expression. TG-treated ARPE19 cells induced the expression of active more than TM-treated casepase-4. The intracellular Ca concentration was elevated in only TG-treated ARPE19 cells. CONCLUSIONS: TG-treated ARPE19 cells showed both Abeta accumulation-inducible and angiogenic factor mRNA expression patterns. This study suggests the possibility that ER stress through ER calcium disruption may induce the expression not only of Abeta deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3-q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

Malfunction of respiratory-related neuronal activity in Na+, K+-ATPase alpha2 subunit-deficient mice is attributable to abnormal Cl- homeostasis in brainstem neurons.

Na+, K+-ATPase 2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2-/-) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2-/- was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2-/- fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2-/- than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2-/-. Lack of inhibitory neural activities in VLM of Atp1a2-/- was observed by optical recordings. High intracellular Cl- concentrations in neurons of the VLM of Atp1a2-/- were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Na+, K+-ATPase alpha2 subunit and KCC2, which excludes Cl- from the cytosol in respiratory center neurons.

Complement C5a receptor-mediated signaling may be involved in neurodegeneration in Alzheimer's disease.

In our earlier results, we demonstrated that cells expressing the complement C5aR are vulnerable since abnormal activation of C5aR caused apoptosis of these cells. In this study, we demonstrate that activation of C5aR by antisense homology box (AHB) peptides synthesized in multiple antigenic peptide form and representing putative interaction sites of the C5a/C5aR evoked calcium influx in TGW neuroblastoma cells. Dose-dependent inhibition of the response was found when the cells were pretreated with C5a, suggesting that C5aR was involved in this process. In addition, pretreatment with monomeric forms of the AHB peptides resulted in attenuation of the calcium signals, supporting the idea of the role of C5aR in this process. Cells of a neuron-rich primary culture and pyramidal cells of rat brain slices also responded to the AHB peptide activation with an increase in the intracellular calcium level, showing that calcium metabolism might be affected in these cells. TUNEL staining demonstrated that C5aR-mediated apoptosis could be induced both in cells of the primary culture as well as in cortical pyramidal neurons of the rat brain. In addition, we investigated expression of C5aR in the hippocampal and cortical neurons of human brains of healthy and demented patients using two anti-human C5aR Abs. Pyramidal cells of the hippocampus and cortex and granular cells of the hippocampus were immunopositive on staining. Although staining was also positive in the vascular dementia brain, it disappeared in the brain with Alzheimer's disease. These results provide further support that C5aR may be involved in neurodegeneration.

Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain.

We investigated the expressions of KCC1, KCC2 and NKCC1 mRNAs in the developing rat brain. The neuroepithelium showed abundant KCC1 and NKCC1 mRNA expressions, while KCC2 mRNA was not detected there. In contrast, KCC2 mRNA was preferentially expressed in postmitotic mature neurons. These results suggest that the appearance of KCC2 expression mainly depends on the maturation of individual neurons.