A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy.

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but also in underlying mechanisms for memory formation such as hippocampal long-term potentiation. Our aim was to identify small molecules with stimulatory effects on the signaling of two neurotrophins, the nerve growth factor (NGF) and the brain derived neurotrophic factor (BDNF). To identify molecules that could potentiate neurotrophin signaling, 25,000 molecules were screened, which led to the identification of the triazinetrione derivatives ACD855 (Ponazuril) and later on ACD856, as positive allosteric modulators of tropomyosin related kinase (Trk) receptors. ACD855 or ACD856 potentiated the cellular signaling of the neurotrophin receptors with EC50 values of 1.9 and 3.2 or 0.38 and 0.30 µM, respectively, for TrkA or TrkB. ACD855 increased acetylcholine levels in the hippocampus by 40% and facilitated long term potentiation in rat brain slices. The compounds acted as cognitive enhancers in a TrkB-dependent manner in several different behavioral models. Finally, the age-induced cognitive dysfunction in 18-month-old mice could be restored to the same level as found in 2-month-old mice after a single treatment of ACD856. We have identified a novel mechanism to modulate the activity of the Trk-receptors. The identification of the positive allosteric modulators of the Trk-receptors might have implications for the treatment of Alzheimer's diseases and other diseases characterized by cognitive impairment.

Human ex vivo spinal cord slice culture as a useful model of neural development, lesion, and allogeneic neural cell therapy.

BACKGROUND: There are multiple promising treatment strategies for central nervous system trauma and disease. However, to develop clinically potent and safe treatments, models of human-specific conditions are needed to complement in vitro and in vivo animal model-based studies. METHODS: We established human brain stem and spinal cord (cross- and longitudinal sections) organotypic cultures (hOCs) from first trimester tissues after informed consent by donor and ethical approval by the Regional Human Ethics Committee, Stockholm (lately referred to as Swedish Ethical Review Authority), and The National Board of Health and Welfare, Sweden. We evaluated the stability of hOCs with a semi-quantitative hOC score, immunohistochemistry, flow cytometry, Ca2+ signaling, and electrophysiological analysis. We also applied experimental allogeneic human neural cell therapy after injury in the ex vivo spinal cord slices. RESULTS: The spinal cord hOCs presented relatively stable features during 7-21 days in vitro (DIV) (except a slightly increased cell proliferation and activated glial response). After contusion injury performed at 7 DIV, a significant reduction of the hOC score, increase of the activated caspase-3+ cell population, and activated microglial populations at 14 days postinjury compared to sham controls were observed. Such elevation in the activated caspase-3+ population and activated microglial population was not observed after allogeneic human neural cell therapy. CONCLUSIONS: We conclude that human spinal cord slice cultures have potential for future structural and functional studies of human spinal cord development, injury, and treatment strategies.

Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor.

The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 µM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor-dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.

Slc7a11 (xCT) protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.

Objectives: The cystine/glutamate antiporter (system xc-) is believed to contribute to nonvesicular glutamate release from glial cells in various brain areas. Although recent investigations implicate system xc- in mood disorders, unambiguous evidence has not yet been established. Therefore, we evaluated the possible role of system xc- in the depressive state. Methods: We conducted a protein expression analysis of the specific subunit of system xc- (xCT) in brain regions of the corticosterone mouse model, Flinders Sensitive Line rat model and post-mortem tissue of depressed patients. We next subjected system xc- deficient mice to the corticosterone model and analysed their behaviour in several tests. Lastly, we subjected additional cohorts of xCT-deficient and wild-type mice to N-acetylcysteine treatment to unveil whether the previously reported antidepressant-like effects are dependent upon system xc-. Results: We did not detect any changes in xCT expression levels in the animal models or patients compared to proper controls. Furthermore, loss of system xc- had no effect on depression- and anxiety-like behaviour. Finally, the antidepressant-like effects of N-acetylcysteine are not mediated via system xc-. Conclusions: xCT protein expression is not altered in the depressed brain and system xc- deficiency does not affect depression-associated behaviour in the corticosterone mouse model.

Aspirin-triggered resolvin D1 prevents surgery-induced cognitive decline.

Hospitalization for major surgery or critical illness often associates with cognitive decline. Inflammation and dysregulation of the innate immune system can exert broad effects in the periphery and central nervous system (CNS), yet the mechanisms underlying memory impairment after surgery remain poorly understood and without effective therapy. Endogenous regulation of acute inflammation is providing novel approaches to treat several disease states including sepsis, pain, obesity and diabetes. Resolvins are potent endogenous lipid mediators biosynthesized during the resolution phase of acute inflammation that display immunoresolvent actions. Here, using a mouse model of surgery-induced cognitive decline we report that orthopedic surgery affects hippocampal neuronal-glial function, including synaptic transmission and plasticity. Systemic prophylaxis with aspirin-triggered resolvin D1 (AT-RvD1: 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, as little as 100 ng dose per mouse) improved memory decline following surgery and abolished signs of synaptic dysfunction. Moreover, delayed administration 24 h after surgery also attenuated signs of neuronal dysfunction postoperatively. AT-RvD1 also limited peripheral damage by modulating the release of systemic interleukin (IL)-6 and improved other clinical markers of tissue injury. Collectively, these results demonstrate a novel role of AT-RvD1 in modulating the proinflammatory milieu after aseptic injury and protecting the brain from neuroinflammation, synaptic dysfunction and cognitive decline. These findings provide novel and safer approaches to treat postoperative cognitive decline and potentially other forms of memory dysfunctions.

Transient calcium and dopamine increase PKA activity and DARPP-32 phosphorylation.

Reinforcement learning theorizes that strengthening of synaptic connections in medium spiny neurons of the striatum occurs when glutamatergic input (from cortex) and dopaminergic input (from substantia nigra) are received simultaneously. Subsequent to learning, medium spiny neurons with strengthened synapses are more likely to fire in response to cortical input alone. This synaptic plasticity is produced by phosphorylation of AMPA receptors, caused by phosphorylation of various signalling molecules. A key signalling molecule is the phosphoprotein DARPP-32, highly expressed in striatal medium spiny neurons. DARPP-32 is regulated by several neurotransmitters through a complex network of intracellular signalling pathways involving cAMP (increased through dopamine stimulation) and calcium (increased through glutamate stimulation). Since DARPP-32 controls several kinases and phosphatases involved in striatal synaptic plasticity, understanding the interactions between cAMP and calcium, in particular the effect of transient stimuli on DARPP-32 phosphorylation, has major implications for understanding reinforcement learning. We developed a computer model of the biochemical reaction pathways involved in the phosphorylation of DARPP-32 on Thr34 and Thr75. Ordinary differential equations describing the biochemical reactions were implemented in a single compartment model using the software XPPAUT. Reaction rate constants were obtained from the biochemical literature. The first set of simulations using sustained elevations of dopamine and calcium produced phosphorylation levels of DARPP-32 similar to that measured experimentally, thereby validating the model. The second set of simulations, using the validated model, showed that transient dopamine elevations increased the phosphorylation of Thr34 as expected, but transient calcium elevations also increased the phosphorylation of Thr34, contrary to what is believed. When transient calcium and dopamine stimuli were paired, PKA activation and Thr34 phosphorylation increased compared with dopamine alone. This result, which is robust to variation in model parameters, supports reinforcement learning theories in which activity-dependent long-term synaptic plasticity requires paired glutamate and dopamine inputs.

DARPP-32 and modulation of cAMP signaling: involvement in motor control and levodopa-induced dyskinesia.

The dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) is abundantly expressed in the medium spiny neurons of the striatum. Phosphorylation catalysed by cAMP-dependent protein kinase (PKA) converts DARPP-32 into an inhibitor of protein phosphatase-1. In contrast, phosphorylation catalysed by cyclin dependent kinase-5 on Thr75 converts DARPP-32 into an inhibitor of PKA. Changes in the state of phosphorylation of DARPP-32 reinforce the behavioral effects produced by stimulation or inhibition of the cAMP pathway. Dopamine, via D(1) receptors, and adenosine, via A(2A) receptors, affect motor behavior by acting on medium spiny neurons, via G(olf) mediated stimulation of the cAMP signaling cascade. The involvement of DARPP-32 in dopamine and adenosine transmission and the possible role played by abnormal regulation of DARPP-32 phosphorylation in levodopa-induced dyskinesia are discussed.

Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum.

The two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), are involved in the control of gene expression via phosphorylation and activation of the transcription factors cyclic AMP response element binding protein (CREB) and Elk-1. Here, we have examined the effect of haloperidol and clozapine, two anti-psychotic drugs, and eticlopride, a selective dopamine D2 receptor antagonist, on the state of phosphorylation of ERK1/2, CREB and Elk-1, in the mouse dorsal striatum. Administration of the typical anti-psychotic haloperidol stimulated the phosphorylation of ERK1/2, CREB and Elk-1. Virtually identical results were obtained using eticlopride. In contrast, the atypical anti-psychotic clozapine reduced ERK1/2, CREB and Elk-1 phosphorylation. This opposite regulation was specifically exerted by haloperidol and clozapine on ERK, CREB, and Elk-1 phosphorylation, as both anti-psychotic drugs increased the phosphorylation of the dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) at the cyclic AMP-dependent protein kinase (PKA) site. The activation of CREB and Elk-1 induced by haloperidol appeared to be achieved via different signalling pathways, as inhibition of ERK1/2 activation abolished the stimulation of Elk-1 phosphorylation without affecting CREB phosphorylation. This study shows that haloperidol and clozapine induce distinct patterns of phosphorylation in the dorsal striatum. The results provide a novel biochemical paradigm elucidating the molecular mechanisms underlying the distinct therapeutic actions of typical and atypical anti-psychotic agents.

Involvement of DARPP-32 phosphorylation in the stimulant action of caffeine.

Caffeine has been imbibed since ancient times in tea and coffee, and more recently in colas. Caffeine owes its psychostimulant action to a blockade of adenosine A(2A) receptors, but little is known about its intracellular mechanism of action. Here we show that the stimulatory effect of caffeine on motor activity in mice was greatly reduced following genetic deletion of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000). Results virtually identical to those seen with caffeine were obtained with the selective A(2A) antagonist SCH 58261. The depressant effect of the A(2A) receptor agonist, CGS 21680, on motor activity was also greatly attenuated in DARPP-32 knockout mice. In support of a role for DARPP-32 in the action of caffeine, we found that, in striata of intact mice, caffeine increased the state of phosphorylation of DARPP-32 at Thr 75. Caffeine increased Thr 75 phosphorylation through inhibition of PP-2A-catalysed dephosphorylation, rather than through stimulation of cyclin-dependent kinase 5 (Cdk5)-catalysed phosphorylation, of this residue. Together, these studies demonstrate the involvement of DARPP-32 and its phosphorylation/dephosphorylation in the stimulant action of caffeine.