Phosphoproteomics implicates glutamatergic and dopaminergic signalling in the antidepressant-like properties of the iron chelator deferiprone.

BACKGROUND: Current antidepressants have limitations due to insufficient efficacy and delay before improvement in symptoms. Polymorphisms of the serotonin transporter (5-HTT) gene have been linked to depression (when combined with stressful life events) and altered response to selective serotonergic reuptake inhibitors. We have previously revealed the antidepressant-like properties of the iron chelator deferiprone in the 5-HTT knock-out (KO) mouse model of depression. Furthermore, deferiprone was found to alter neural activity in the prefrontal cortex of both wild-type (WT) and 5-HTT KO mice. METHODS: In the current study, we examined the molecular effects of acute deferiprone treatment in the prefrontal cortex of both genotypes via phosphoproteomics analysis. RESULTS: In WT mice treated with deferiprone, there were 22 differentially expressed phosphosites, with gene ontology analysis implicating cytoskeletal proteins. In 5-HTT KO mice treated with deferiprone, we found 33 differentially expressed phosphosites. Gene ontology analyses revealed phosphoproteins that were predominantly involved in synaptic and glutamatergic signalling. In a drug-naïve cohort (without deferiprone administration), the analysis revealed 21 differentially expressed phosphosites in 5-HTT KO compared to WT mice. We confirmed the deferiprone-induced increase in tyrosine hydroxylase serine 40 residue phosphorylation (pTH-Ser40) (initially revealed in our phosphoproteomics study) by Western blot analysis, with deferiprone increasing pTH-Ser40 expression in WT and 5-HTT KO mice. CONCLUSION: As glutamatergic and synaptic signalling are dysfunctional in 5-HTT KO mice (and are the target of fast-acting antidepressant drugs such as ketamine), these molecular effects may underpin deferiprone's antidepressant-like properties. Furthermore, dopaminergic signalling may also be involved in deferiprone's antidepressant-like properties.

Perturbed iron biology in the prefrontal cortex of people with schizophrenia.

Despite loss of grey matter volume and emergence of distinct cognitive deficits in young adults diagnosed with schizophrenia, current treatments for schizophrenia do not target disruptions in late maturational reshaping of the prefrontal cortex. Iron, the most abundant transition metal in the brain, is essential to brain development and function, but in excess, it can impair major neurotransmission systems and lead to lipid peroxidation, neuroinflammation and accelerated aging. However, analysis of cortical iron biology in schizophrenia has not been reported in modern literature. Using a combination of inductively coupled plasma-mass spectrometry and western blots, we quantified iron and its major-storage protein, ferritin, in post-mortem prefrontal cortex specimens obtained from three independent, well-characterised brain tissue resources. Compared to matched controls (n = 85), among schizophrenia cases (n = 86) we found elevated tissue iron, unlikely to be confounded by demographic and lifestyle variables, by duration, dose and type of antipsychotic medications used or by copper and zinc levels. We further observed a loss of physiologic age-dependent iron accumulation among people with schizophrenia, in that the iron level among cases was already high in young adulthood. Ferritin, which stores iron in a redox-inactive form, was paradoxically decreased in individuals with the disorder. Such iron-ferritin uncoupling could alter free, chemically reactive, tissue iron in key reasoning and planning areas of the young-adult schizophrenia cortex. Using a prediction model based on iron and ferritin, our data provide a pathophysiologic link between perturbed cortical iron biology and schizophrenia and indicate that achievement of optimal cortical iron homeostasis could offer a new therapeutic target.

In Vivo 7-Tesla MRI Investigation of Brain Iron and Its Metabolic Correlates in Chronic Schizophrenia.

Brain iron is central to dopaminergic neurotransmission, a key component in schizophrenia pathology. Iron can also generate oxidative stress, which is one proposed mechanism for gray matter volume reduction in schizophrenia. The role of brain iron in schizophrenia and its potential link to oxidative stress has not been previously examined. In this study, we used 7-Tesla MRI quantitative susceptibility mapping (QSM), magnetic resonance spectroscopy (MRS), and structural T1 imaging in 12 individuals with chronic schizophrenia and 14 healthy age-matched controls. In schizophrenia, there were higher QSM values in bilateral putamen and higher concentrations of phosphocreatine and lactate in caudal anterior cingulate cortex (caCC). Network-based correlation analysis of QSM across corticostriatal pathways as well as the correlation between QSM, MRS, and volume, showed distinct patterns between groups. This study introduces increased iron in the putamen in schizophrenia in addition to network-wide disturbances of iron and metabolic status.

The ubiquitin proteasome system and schizophrenia.

The ubiquitin-proteasome system is a master regulator of neural development and the maintenance of brain structure and function. It influences neurogenesis, synaptogenesis, and neurotransmission by determining the localisation, interaction, and turnover of scaffolding, presynaptic, and postsynaptic proteins. Moreover, ubiquitin-proteasome system signalling transduces epigenetic changes in neurons independently of protein degradation and, as such, dysfunction of components and substrates of this system has been linked to a broad range of brain conditions. Although links between ubiquitin-proteasome system dysfunction and neurodegenerative disorders have been known for some time, only recently have similar links emerged for neurodevelopmental disorders, such as schizophrenia. Here, we review the components of the ubiquitin-proteasome system that are reported to be dysregulated in schizophrenia, and discuss specific molecular changes to these components that might, in part, explain the complex causes of this mental disorder.

Blood and brain protein levels of ubiquitin-conjugating enzyme E2K (UBE2K) are elevated in individuals with schizophrenia.

A number of recent studies have suggested the ubiquitin proteasome system (UPS) in schizophrenia is dysfunctional. The purpose of this study was to investigate UBE2K, a ubiquitin-conjugating (E2) enzyme within the UPS that has been associated with psychosis symptom severity, in the blood and brain of individuals with schizophrenia. Whole blood and erythrocytes from 128 (71 treatment-resistant schizophrenia, 57 healthy controls) individuals as well as frozen dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC) post-mortem samples from 74 (37 schizophrenia, 37 controls) individuals were obtained. UBE2K gene expression was assayed in whole blood and DLPFC samples, whereas protein levels were assayed in erythrocytes and OFC samples. Elevated levels of UBE2K mRNA were observed in whole blood of individuals with schizophrenia (p = 0.03) but not in the DLPFC, while protein levels were raised in erythrocytes and the OFC (p < 0.001 and p = 0.002 respectively). Findings were not better explained by age, smoking, clozapine plasma levels or duration of illness. Although blood and brain samples were derived from independent samples, our findings suggest peripheral protein levels of UBE2K may serve as a surrogate of brain levels and further supports the notion of UPS dysfunction in schizophrenia. Future studies to determine the pathophysiological effects of elevated UBE2K protein levels in the brain of those with schizophrenia are warranted.

Elevated ubiquitinated proteins in brain and blood of individuals with schizophrenia.

Dysregulation of the ubiquitin proteasome system (UPS) has been linked to schizophrenia but it is not clear if this dysregulation is detectable in both brain and blood. We examined free mono-ubiquitin, ubiquitinated proteins, catalytic ubiquitination, and proteasome activities in frozen postmortem OFC tissue from 76 (38 schizophrenia, 38 control) matched individuals, as well as erythrocytes from 181 living participants, who comprised 30 individuals with recent onset schizophrenia (mean illness duration = 1 year), 63 individuals with 'treatment-resistant' schizophrenia (mean illness duration = 17 years), and 88 age-matched participants without major psychiatric illness. Ubiquitinated protein levels were elevated in postmortem OFC in schizophrenia compared to controls (p = <0.001, AUC = 74.2%). Similarly, individuals with 'treatment-resistant' schizophrenia had higher levels of ubiquitinated proteins in erythrocytes compared to those with recent onset schizophrenia (p < 0.001, AUC = 65.5%) and controls (p < 0.001, AUC = 69.4%). The results could not be better explained by changes in proteasome activity, demographic, medication, or tissue factors. Our results suggest that ubiquitinated protein formation may be abnormal in both the brain and erythrocytes of those with schizophrenia, particularly in the later stages or specific sub-groups of the illness. A derangement in protein ubiquitination may be linked to pathogenesis or neurotoxicity in schizophrenia, and its manifestation in the blood may have prognostic utility.

Copper-uptake is critical for the down regulation of synapsin and dynamin induced by neocuproine: modulation of synaptic activity in hippocampal neurons.

Extracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin-3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer's and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.

Biphasic effects of copper on neurotransmission in rat hippocampal neurons.

The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5 µM; copper: EC50 2.9 ± 0.2 µM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).

Actinic cheilitis: epithelial expression of COX-2 and its association with mast cell tryptase and PAR-2.

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies, including oral cancers. Recent studies have shown that mast cell-derived protease tryptase can induce COX-2 expression by the cleavage of proteinase-activated receptor-2 (PAR-2). Actinic cheilitis (AC) is a premalignant form of lip cancer characterized by an increased density of tryptase-positive mast cells. To investigate the possible contribution of tryptase to COX-2 overexpression during early lip carcinogenesis, normal lip (n=24) and AC (n=45) biopsies were processed for COX-2, PAR-2 and tryptase detection, using RT-PCR and immunohistochemistry. Expression scores were obtained for each marker and tested for statistical significance using Mann-Whitney and Spearmann's correlation tests as well as multivariate logistic regression analysis. Increased epithelial co-expression of COX-2 and PAR-2, as well as, elevated subepithelial density of tryptase-positive mast cells were found in AC as compared to normal lip (P<0.001). COX-2 overexpression was found to be a significant predictor of AC (P<0.034, forward stepwise, Wald), and to be correlated with both tryptase-positive mast cells and PAR-2 expression (P<0.01). The results suggest that epithelial COX-2 overexpression is a key event in AC, which is associated with increased tryptase-positive mast cells and PAR-2. Therefore, tryptase may contribute to COX-2 up-regulation by epithelial PAR-2 activation during early lip carcinogenesis.

The influence of mast cell mediators on migration of SW756 cervical carcinoma cells.

The role of mast cell mediators on cervical cancer cell migration was assessed using an in vitro assay of scratch wound healing onto monolayers of HPV18-positive cervical carcinoma cells (SW756). Migration of SW756 cells was accelerated by co-culture with the mast cell line LAD2. This effect was inhibited by the H1R antagonist pyrilamine and the cannabinoid agonists 2-arachidonylglycerol (2AG) and Win 55,212-2. Therefore, the specific effects of histamine and cannabinoids on SW756 migration and LAD2 activation were analyzed. Histamine added to the in vitro assay of scratch wound healing either increased or inhibited SW756 migration rate by acting either on H1R or H4R, respectively. Cannabinoids acted on CB1 receptors to inhibit SW756 migration. Supernatants from SW756 cells stimulated LAD2 cell degranulation, which in turn was inhibited by cannabinoids acting via CB2 receptors. RT-PCR showed that SW756 expressed mRNA for CB1, CB2, H1R, H2R, and H4R. On the other hand, LAD2 expressed mRNA for all four HRs and CB2. The results suggest that mast cells could be contributing to cervical cancer cell invasion and spreading by the release of histamine and cannabinoids. Therefore, therapeutic modulation of specific mast cell mediators may be beneficial for cervical cancer treatment.

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease.

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.