Detection of 14-3-3ζ in cerebrospinal fluid following experimentally evoked seizures

Surrogate and peripheral (bio)markers of neuronal injury may be of value in assessing effects of seizures on the brain or epilepsy development following trauma. The presence of 14-3-3 isoforms in cerebrospinal fluid (CSF) is a diagnostic indicator of Creutzfeldt–Jakob disease but these proteins may also be present following acute neurological insults. Here, we examined neuronal and 14-3-3 proteins in CSF from rats after seizures. Seizures induced by intra-amygdala microinjection of 0.1 µg kainic acid (KA) caused damage which was mainly restricted to the ipsilateral CA3 subfield of the hippocampus. 14-3-3ζ was detected at significant levels in CSF sampled 4 h after seizures compared with near absence in control CSF. Neuron-specific nuclear protein (NeuN) was also elevated in CSF in seizure rats. CSF 14-3-3ζ levels were significantly lower in rats treated with 0.01 µg KA. These data suggest the presence of 14-3-3ζ within CSF may be a biomarker of acute seizure damage.     

Depletion of 14-3-3 zeta elicits endoplasmic reticulum stress and cell death, and increases vulnerability to kainate-induced injury in mouse hippocampal cultures

14-3-3 proteins are ubiquitous signalling molecules that regulate development and survival pathways in brain. Altered expression and cellular localization of 14-3-3 proteins has been implicated in neurodegenerative diseases and in neuronal death after acute neurological insults, including seizures. Presently, we examined expression and function of 14-3-3 isoforms in vitro using mouse organotypic hippocampal cultures. Treatment of cultures with the endoplasmic reticulum (ER) stressor tunicamycin caused an increase in levels of 14-3-3 zeta within the ER-containing microsomal fraction, along with up-regulation of Lys-Asp-Glu-Leu-containing proteins and calnexin, and the selective death of dentate granule cells. Depletion of 14-3-3 zeta levels using small interfering RNA induced both ER stress proteins and death of granule cells. Treatment of hippocampal cultures with the excitotoxin kainic acid increased levels of Lys-Asp-Glu-Leu-containing proteins and microsomal 14-3-3 zeta levels and caused cell death within the CA1, CA3 and dentate gyrus of the hippocampus. Kainic acid-induced damage was significantly increased in each hippocampal subfield of cultures treated with small interfering RNA targeting 14-3-3 zeta. The present data indicate a role for 14-3-3 zeta in survival responses following ER stress and possibly protection against seizure injury to the hippocampus.     

Ubiquitin Carboxy-Terminal Hydrolase L1 (UCH-L1) is increased in cerebrospinal fluid and plasma of patients after epileptic seizure

ABSTRACT: BACKGROUND: Clinical and experimental studies have demonstrated that seizures can cause molecular and cellular responses resulting in neuronal damage. At present, there are no valid tests for assessing organic damage to the brain associated with seizure. The aim of this study was to investigate cerebrospinal fluid (CSF) and plasma concentrations of Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a sensitive indicator of acute injury to brain neurons, in patients with tonic--clonic or partial secondarily generalized seizures due to various etiologies. METHODS: CSF and plasma concentrations of UCH-L1 were assessed in 52 patients within 48 hours after epileptic seizure and in 19 controls using ELISA assays. RESULTS: CSF obtained within 48 hours after seizure or status epilepticus (SE) presented significantly higher levels of UCH-L1 compared to controls (p = 0.008). Plasma UCH-L1 concentrations were negatively correlated with time to sample withdrawal. An analysis conducted using only the first 12 hours post-seizure revealed significant differences between concentrations of UCH-L1 in plasma and controls (p = 0.025). CSF and plasma concentrations were strongly correlated with age in patients with seizure, but not in control patients. Plasma UCH-L1 levels were also significantly higher in patients after recurrent seizures (n = 4) than in those after one or two seizures (p = 0.013 and p = 0.024, respectively). CONCLUSION: Our results suggest that determining levels of neuronal proteins may provide valuable information on the assessment of brain damage following seizure. These data might allow clinicians to make more accurate therapeutic decisions, to identify patients at risk of progression and, ultimately, to provide new opportunities for monitoring therapy and targeted therapeutic interventions.     

Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3gamma and -zeta were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3gamma levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3zeta, while cytoplasm-localized 14-3-3beta, -epsilon and -eta remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3epsilon and -zeta levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures.     

Interaction of 14-3-3 with Bid during seizure-induced neuronal death

Seizure-induced neuronal death may involve coordinated intracellular trafficking and protein-protein interactions of members of the Bcl-2 family. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3-interacting domain death agonist (Bid) may contribute to seizure-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid during seizure-induced neuronal death. Brief seizures were evoked in rats by intraamygdala microinjection of kainic acid to elicit unilateral hippocampal CA3 neuronal death. Coimmunoprecipitation analysis demonstrated that although Bcl-2-associated death promoter (Bad) constitutively bound 14-3-3, there was no interaction between Bid and 14-3-3 in control brain. Seizures triggered Bid cleavage and a commensurate increase in binding of Bid to 14-3-3 within injured hippocampus. Casein kinases I and II, which can inactivate Bid by phosphoserine/threonine modification, did not coimmunoprecipitate with Bid. The largely uninjured contralateral hippocampus did not exhibit Bid cleavage or binding of 14-3-3 to Bid. In vitro experiments confirmed that 14-3-3beta is capable of binding truncated Bid, likely in the absence of phosphoserine/threonine modification. These data suggest 14-3-3 proteins may target active as well as inactive conformations of pro-apoptotic Bcl-2 death agonists, highlighting novel targets for intervention in seizure-induced neuronal death.     

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.     

Specific 14-3-3 isoform detection and immunolocalization in prion diseases

14-3-3 proteins are involved in signalling processes in neuronal cells. Using isoform-specific antibodies we have examined the variation in 14-3-3 isoform neurolocation in normal and scrapie-infected murine brain and show that in defined areas of the brain there are significant changes associated with the pathology of the disease process. The appearance of 14-3-3 proteins in the cerebrospinal fluid (CSF) is a consequence of neuronal disease and the detection of specific isoforms of the 14-3-3 proteins in the CSF is characteristic of some neurodegenerative diseases. In this study, monitoring specifically for the gamma 14-3-3 isoform in the CSF by both Western-blot analysis and ELISA we can show a level of correlation between the assays.     

14-3-3 regulates actin dynamics by stabilizing phosphorylated cofilin

The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles. Rho GTPases act through p21-activated kinase-1 (Pak-1) and Rho kinase to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Ser3. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Ser3 phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Ser3 phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3zeta increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Ser3 by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.     

Identification and characterization of the interaction between tuberin and 14-3-3zeta

Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called tuberin. Although it has been shown that hamartin and tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with tuberin. One of the proteins identified was 14-3-3zeta, a member of the 14-3-3 protein family. The interaction between tuberin and 14-3-3zeta was confirmed in vitro and by co-immunoprecipitation; multiple sites within tuberin for 14-3-3zeta binding were identified; and it was determined that 14-3-3zeta associated with the tuberin-hamartin complex. Finally, it was shown that the tuberin/14-3-3zeta interaction is regulated by Akt-mediated phosphorylation of tuberin, providing insight into how tuberin may regulate phosphorylation of S6.     

Anticonvulsant properties of the cerebrospinal fluid during activation of the antiepileptic system of the brain

Cerebrospinal fluid (CSF) was obtained after 30-40 sessions of daily electrical stimulation of the cat cerebellum vermis. The intraventricular injection of CSF (10 microliters) to Wistar rats increased the latent period of initial seizure manifestations, significantly reduced the number of animals with seizures and reduced the severity of seizures induced by korazol injection (40 mg/kg). Analogous seizure changes were observed in rats after intraventricular injection of CSF (10 microliters) from cats subject to 3-10 electroshock seizure fits. Intraventricular injection of CSF (250 microliters) obtained from cats after electroshock to cats with strychnine-induced epileptic foci in the brain cortex led to the suppression of the epileptic activity. The conclusion was made that different ways of antiepileptic system activation cause the accumulation of endogenous antiepileptic substances in CSF.     

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II–IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3zeta and Bm14-3-3epsilon). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3zeta shared 90% identity with that in Drosophila, while Bm14-3-3epsilon shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3zeta expression levels are higher than Bm14-3-3epsilon in seven tissues and in four silkworm developmental stages examined. Bm14-3-3zeta was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3epsilon expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3zeta expression is universal and continuous, while Bm14-3-3epsilon expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures

The cysteine protease caspase-3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure-induced changes in caspase-3 protein expression and processing, enzyme activity, and the in vivo effect of caspase-3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 microg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0-72 h following diazepam administration. Levels of the 32-kDa proenzyme form of caspase-3 were unaffected by seizures. Levels of the 17-kDa cleaved (active) fragment of caspase-3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase-3-like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase-3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase-3 may play a significant role in the mechanism by which neurons die following seizures.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

MADM,a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.     

Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation

The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, beta, epsilon, theta and zeta, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, sigma and gamma, were present only at very low levels. Immunoprecipitation data showed 14-3-3zeta could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3zeta in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G(2)/M-phase arrest. Moreover, 16% 14-3-3zeta antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3zeta is involved in G(2) checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.     

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

Background

Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ_1–42 were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed.

Methods

CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients.

Results

We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP^c) and Aβ_1–42 levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions.

Conclusion

Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins.     

Characterization of 14-3-3sigma Dimerization Determinants: Requirement of Homodimerization for Inhibition of Cell Proliferation

The seven highly conserved 14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- and hetero-dimers, which is poorly characterized. Among the 14-3-3 proteins 14-3-3sigma is unique as it has tumor suppressive properties. Expression of 14-3-3sigma is induced by DNA damage in a p53-dependent manner and mediates a cell cycle arrest. Here we show that the 14-3-3sigma protein exclusively forms homodimers when it is ectopically expressed at high levels, whereas ectopic 14-3-3zeta formed heterodimers with the 5 other 14-3-3 isoforms. The x-ray structure of 14-3-3sigma?revealed 5 residues (Ser⁵, Glu²⁰, Phe²⁵, Q⁵⁵, Glu⁸⁰) as candidate determinants of dimerization specificity. Here we converted these amino-acids to residues present in 14-3-3zeta at the analogous positions. Thereby, Ser⁵, Glu²⁰ and Glu⁸⁰ were identified as key residues responsible for the selective homodimerization of 14-3-3sigma. Conversion of all 5 candidate residues was sufficient to switch the dimerization pattern of 14-3-3sigma to a pattern which is very similar to that of 14-3-3zeta. In contrast to wildtype 14-3-3sigma this 14-3-3sigma variant and 14-3-3zeta were unable to mediate inhibition of cell proliferation. Therefore, homodimerization by 14-3-3sigma is required for its unique functions among the 7 mammalian 14-3-3 proteins. As inactivation of 14-3-3sigma sensitizes to DNA-damaging drugs, substances designed to interfere with 14-3-3sigma dimerization may be used to inactivate 14-3-3sigma function for cancer therapeutic purposes.