CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures.

BAR (Bin, Amphiphysin, and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse BAR) domain containing tools derived from the fusion of the Arabidopsis thaliana cryptochrome 2 photoreceptor and I-BAR protein domains ("CRY-BARs") with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane-binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and phosphatidylinositol 4,5-bisphosphate-binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Prenatal and postnatal genetic testing toward personalized care: The non-invasive perinatal testing.

This article investigates how non-invasive prenatal testing and the incorporation of genomic sequencing into newborn screening postnatally are transforming perinatal care. They improve the accuracy of prenatal and neonatal screening, allowing for early interventions and personalized therapies. Non-invasive prenatal testing before birth and saliva-sample-based newborn genomic sequencing after birth can be collectively referred to as non-invasive perinatal testing. Non-invasive prenatal testing is particularly useful for aneuploidy, whereas performance markers worsen as DNA abnormalities shrink in size. Screening for clinically actionable diseases in childhood would be crucial to personalized medical therapy, as the postnatal period remains appropriate for screening for the great majority of monogenic disorders. While genomic data can help diagnose uncommon diseases, challenges like ethics and equity necessitate joint approaches for appropriate integration in this revolutionary journey toward personalized care.

CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells.

The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as ß-amyloid (Aß) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.

Activation of CaMKII in single dendritic spines during long-term potentiation.

Calcium/calmodulin-dependent kinase II (CaMKII) plays a central part in long-term potentiation (LTP), which underlies some forms of learning and memory. Here we monitored the spatiotemporal dynamics of CaMKII activation in individual dendritic spines during LTP using two-photon fluorescence lifetime imaging microscopy, in combination with two-photon glutamate uncaging. Induction of LTP and associated spine enlargement in single spines triggered transient ( approximately 1 min) CaMKII activation restricted to the stimulated spines. CaMKII in spines was specifically activated by NMDA receptors and L-type voltage-sensitive calcium channels, presumably by nanodomain Ca(2+) near the channels, in response to glutamate uncaging and depolarization, respectively. The high degree of compartmentalization and channel specificity of CaMKII signalling allow stimuli-specific spatiotemporal patterns of CaMKII signalling and may be important for synapse-specificity of synaptic plasticity.

Centaurin-α1-Ras-Elk-1 signaling at mitochondria mediates ß-amyloid-induced synaptic dysfunction.

Alzheimer's disease is thought to be caused by ß-amyloid peptide (Aß)-dependent synaptic dysfunction. However, the signaling pathways connecting Aß and synaptic dysfunction remain elusive. Here we report that Aß transiently increases the expression level of centaurin-α1 (CentA1) in neurons, which induces a Ras-dependent association of Elk-1 with mitochondria, leading to mitochondrial and synaptic dysfunction in organotypic hippocampal slices of rats. Downregulation of the CentA1-Ras-Elk-1 pathway restored normal mitochondrial activity, spine structural plasticity, spine density, and the amplitude and frequency of miniature EPSCs in Aß-treated neurons, whereas upregulation of the pathway was sufficient to decrease spine density. Elevations of CentA1 and association of Elk-1 with mitochondria were also observed in transgenic mice overexpressing a human mutant form of amyloid precursor protein. Therefore, the CentA1-Ras-Elk-1 signaling pathway acts on mitochondria to regulate dendritic spine density and synaptic plasticity in response to Aß in hippocampal neurons, providing new pharmacological targets for Alzheimer's disease.

AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK-dependent manner during long-term potentiation.

The exocytosis of AMPA receptors is a key step in long-term potentiation (LTP), yet the timing and location of exocytosis and the signaling pathways involved in exocytosis during synaptic plasticity are not fully understood. Here we combine two-photon uncaging with two-photon imaging of a fluorescent label of surface AMPA receptors to monitor individual AMPA receptor exocytosis events near spines undergoing LTP. AMPA receptors that reached the stimulated spine came from a combination of preexisting surface receptors (70-90%) and newly exocytosed receptors (10-30%). We observed exocytosis in both the dendrite and spine under basal conditions. The rate of AMPA receptor exocytosis increased approximately 5-fold during LTP induction and decayed to the basal level within approximately 1 min, both in the stimulated spine and in the dendrite within approximately 3 microm of the stimulated spine. AMPA receptors inserted in the spine were trapped in the spine in an activity-dependent manner. The activity-dependent exocytosis required the Ras-ERK pathway, but not CaMKII. Thus, diffusive Ras-ERK signaling presumably serves as an important means for signaling from synapses to dendritic shafts to recruit AMPA receptors into synapses during LTP.

[Giant uterine leiomyoma causing acute symptoms]. / Akut tüneteket okozó óriásmyoma.

The case history of a 71-year-old woman with acute dyspnoea caused by a giant leiomyoma and severe acute anemia due to intratumoral hemorrhage is presented. Urgent operation was performed, and a 13.5 kg pendular tumor was removed. The cornerstones of the differential diagnoses and therapy of giant abdominal tumors is discussed.

[Methods and conditions of fertility preservation in early-stage ovarian tumors]. / A fertilitás megtartásának lehetoségei, feltételei és módjai korai petefészek-daganatok esetén.

The aim of this study is to review the literature of fertility-sparing techniques and their safety in early-stage malignant ovarian tumors, especially in epithelial ovarian cancer. Fertility preservation is widely accepted in early-stage borderline, germ cell and sex cord-stromal tumors. Based on data from retrospective studies, fertility-sparing surgery in epithelial ovarian cancer can be recommended in stage IA, grade 1-2 and favorable hystologic type ovarian cancer. Above stage IA, or in grade 3, or in clear-cell tumors decision making process about fertility-sparing surgery should be individual. Correct surgical staging is mandatory and oncologic safety should be of primary importance. In the group of carefully selected patients oncological outcomes are identical to those of radical surgery. Spontaneous pregnancy rates vary, but they are generally high. Adequate counseling with patients, detailed documentation and careful follow-up is of outstanding importance. In order to improve the quality of fertility preservation techniques, establishment of treatment centers is recommended.

Crosstalk between the Rho and Rab family of small GTPases in neurodegenerative disorders.

Neurodegeneration is associated with defects in cytoskeletal dynamics and dysfunctions of the vesicular trafficking and sorting systems. In the last few decades, studies have demonstrated that the key regulators of cytoskeletal dynamics are proteins from the Rho family GTPases, meanwhile, the central hub for vesicle sorting and transport between target membranes is the Rab family of GTPases. In this regard, the role of Rho and Rab GTPases in the induction and maintenance of distinct functional and morphological neuronal domains (such as dendrites and axons) has been extensively studied. Several members belonging to these two families of proteins have been associated with many neurodegenerative disorders ranging from dementia to motor neuron degeneration. In this analysis, we attempt to present a brief review of the potential crosstalk between the Rab and Rho family members in neurodegenerative pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, and amyotrophic lateral sclerosis (ALS).

Behavioral and Transcriptome Profiling of Heterozygous Rab10 Knock-Out Mice.

A central question in the field of aging research is to identify the cellular and molecular basis of neuroresilience. One potential candidate is the small GTPase, Rab10. Here, we used Rab10+/- mice to investigate the molecular mechanisms underlying Rab10-mediated neuroresilience. Brain expression analysis of 880 genes involved in neurodegeneration showed that Rab10+/- mice have increased activation of pathways associated with neuronal metabolism, structural integrity, neurotransmission, and neuroplasticity compared with their Rab10+/+ littermates. Lower activation was observed for pathways involved in neuroinflammation and aging. We identified and validated several differentially expressed genes (DEGs), including Stx2, Stx1b, Vegfa, and Lrrc25 (downregulated) and Prkaa2, Syt4, and Grin2d (upregulated). Behavioral testing showed that Rab10+/- mice perform better in a hippocampal-dependent spatial task (object in place test), while their performance in a classical conditioning task (trace eyeblink classical conditioning, TECC) was significantly impaired. Therefore, our findings indicate that Rab10 differentially controls the brain circuitry of hippocampal-dependent spatial memory and higher-order behavior that requires intact cortex-hippocampal circuitry. Transcriptome and biochemical characterization of these mice suggest that glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D or GluN2D) is affected by Rab10 signaling. Further work is needed to evaluate whether GRIN2D mediates the behavioral phenotypes of the Rab10+/- mice. We conclude that Rab10+/- mice described here can be a valuable tool to study the mechanisms of resilience in Alzheimer's disease (AD) model mice and to identify novel therapeutical targets to prevent cognitive decline associated with normal and pathologic aging.

Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures.

Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer's Disease (AD). We have recently developed a novel optically-responsive tool (the 'CofActor' system) that couples cof ilin and act in (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a light-activated, genetically encoded redox sensor that can be activated with precise spatial and temporal control. Here we describe a protocol for the expression and activation of the CofActor system in dissociated hippocampal neuron cultures prepared from newborn mice. Cultures were transfected with Lipofectamine on the fifth day in vitro (DIV5), then exposed to cellular stress inducing stimuli, leading to the formation of actin-cofilin rods that can be observed using live cell imaging techniques. The protocol described here allows for studies of stress-related cytoskeletal dysregulation in live neurons exposed to neurodegenerative stimuli, such as toxic Aß42 oligomers. Moreover, expression of the sensor in neurons isolated from transgenic mouse models of AD and/or mice KO for proteins involved in AD can advance our understanding of the molecular basis of early cytoskeletal dysfunctions associated with neurodegeneration.

ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus.

ADAP1/Centaurin-α1 (CentA1) functions as an Arf6 GTPase-activating protein highly enriched in the brain. Previous studies demonstrated the involvement of CentA1 in brain function as a regulator of dendritic differentiation and a potential mediator of Alzheimer's disease (AD) pathogenesis. To better understand the neurobiological functions of CentA1 signaling in the brain, we developed Centa1 knock-out (KO) mice. The KO animals showed neither brain development nor synaptic ultrastructure deficits in the hippocampus. However, they exhibited significantly higher density and enhanced structural plasticity of dendritic spines in the CA1 region of the hippocampus compared with non-transgenic (NTG) littermates. Moreover, the deletion of Centa1 improved performance in the object-in-place (OIP) spatial memory task. These results suggest that CentA1 functions as a negative regulator of spine density and plasticity, and of hippocampus-dependent memory formation. Thus, CentA1 and its downstream signaling may serve as a potential therapeutic target to prevent memory decline associated with aging and brain disorders.

Role of kinase suppressor of Ras-1 in neuronal survival signaling by extracellular signal-regulated kinase 1/2.

Scaffolding proteins including kinase suppressor of Ras-1 (KSR1) determine specificity of signaling by extracellular signal-regulated kinase 1/2 (ERK1/2), enabling it to couple diverse extracellular stimuli to various cellular responses. The scaffolding protein(s) that contributes to ERK1/2-mediated neuronal survival has not yet been identified. In cultured rat cortical neurons, BDNF activates ERK1/2 to enhance neuronal survival by suppressing DNA damage- or trophic deprivation-induced apoptosis. Here we report that in this system, BDNF increased KSR1 association with activated ERK1/2, whereas KSR1 knockdown with a short hairpin (sh) RNA reduced BDNF-mediated activation of ERK1/2 and protection against a DNA-damaging drug, camptothecin (CPT). In contrast, BDNF suppression of trophic deprivation-induced apoptosis was unaffected by shKSR1 although blocked by shERK1/2. Also, overexpression of KSR1 enhanced BDNF protection against CPT. Therefore, KSR1 is specifically involved in antigenotoxic activation of ERK1/2 by BDNF. To test whether KSR1 contributes to ERK1/2 activation by other neuroprotective stimuli, we used a cAMP-elevating drug, forskolin. In cortical neurons, ERK1/2 activation by forskolin was protein kinase A (PKA) dependent but TrkB (receptor tyrosine kinase B) independent and was accompanied by the increased association between KSR1 and active ERK1/2. Forskolin suppressed CPT-induced apoptosis in a KSR1 and ERK1/2-dependent manner. Inhibition of PKA abolished forskolin protection, whereas selective PKA activation resulted in an ERK1/2- and KSR1-mediated decrease in apoptosis. Hence, KSR1 is critical for the antiapoptotic activation of ERK1/2 by BDNF or cAMP/PKA signaling. In addition, these novel data indicate that stimulation of cAMP signaling is a candidate neuroprotective strategy to intervene against neurotoxicity of DNA-damaging agents.

Cisplatin-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases.

The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.

CaMKII Autophosphorylation Is Necessary for Optimal Integration of Ca2+ Signals during LTP Induction, but Not Maintenance.

CaMKII plays a critical role in decoding calcium (Ca2+) signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ signals in spines remain elusive. Here, we measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each pulse during repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. Thr286 phosphorylation slows the decay of CaMKII and thus lowers the frequency required to induce spine plasticity by several fold. In the absence of Thr286 phosphorylation, increasing the stimulation frequency results in high peak mutant CaMKIIT286A activity that is sufficient for inducing plasticity. Our findings demonstrate that Thr286 phosphorylation plays an important role in induction of LTP by integrating Ca2+ signals, and it greatly promotes, but is dispensable for, the activation of CaMKII and LTP.

Kinetics of Endogenous CaMKII Required for Synaptic Plasticity Revealed by Optogenetic Kinase Inhibitor.

Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.

Blood-brain barrier changes during compensated and decompensated hemorrhagic shock.

Dysfunction of the blood-brain barrier (BBB) can be associated with a large number of central nervous system and systemic disorders. The aim of the present study was to determine BBB changes during different phases of hemorrhagic shock. The experiments were carried out on male Wistar rats anaesthetized with urethane. To produce compensated or decompensated hemorrhagic shock, mean arterial pressure was decreased from the normotensive control values to 40 mmHg by a standardized method of blood withdrawal from the femoral artery. Cerebral blood flow changes were followed by laser-Doppler flowmetry, and arterial blood gas values were monitored over the whole procedure. Cortical blood flow was significantly reduced in compensated and in decompensated hemorrhagic shock compared with the normotensive rats. As the shock shifted to the decompensated phase, the blood flow reduction was more pronounced. BBB permeability studies using sodium fluorescein (molecular weight of 376) and Evan's Blue albumin (molecular weight of 67,000) have revealed a significant increase of the BBB permeability for sodium fluorescein in the decompensated stage of hemorrhagic shock. Western blot analysis of brain capillaries showed that the expression of the transmembrane tight junction protein occludin was reduced in response to hemorrhagic shock, and the decrease of occludin was more pronounced in the decompensated phase. A similar expression pattern was shown by the transmembrane adherens junction protein cadherin as well. Our results suggest that the decompensated phase of hemorrhagic shock is associated with disturbances of the BBB, which may be explained by the dysfunction of interendothelial junctions caused by decreased occludin and cadherin levels.

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione.

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.

NMDA neuroprotection against a phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of glycogen synthase kinase-3beta-induced apoptosis.

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.