Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

Glutamate exocytosis from astrocytes controls synaptic strength.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

Beyond intracranial pressure: optimization of cerebral blood flow, oxygen, and substrate delivery after traumatic brain injury.

Monitoring and management of intracranial pressure (ICP) and cerebral perfusion pressure (CPP) is a standard of care after traumatic brain injury (TBI). However, the pathophysiology of so-called secondary brain injury, i.e., the cascade of potentially deleterious events that occur in the early phase following initial cerebral insult-after TBI, is complex, involving a subtle interplay between cerebral blood flow (CBF), oxygen delivery and utilization, and supply of main cerebral energy substrates (glucose) to the injured brain. Regulation of this interplay depends on the type of injury and may vary individually and over time. In this setting, patient management can be a challenging task, where standard ICP/CPP monitoring may become insufficient to prevent secondary brain injury. Growing clinical evidence demonstrates that so-called multimodal brain monitoring, including brain tissue oxygen (PbtO2), cerebral microdialysis and transcranial Doppler among others, might help to optimize CBF and the delivery of oxygen/energy substrate at the bedside, thereby improving the management of secondary brain injury. Looking beyond ICP and CPP, and applying a multimodal therapeutic approach for the optimization of CBF, oxygen delivery, and brain energy supply may eventually improve overall care of patients with head injury. This review summarizes some of the important pathophysiological determinants of secondary cerebral damage after TBI and discusses novel approaches to optimize CBF and provide adequate oxygen and energy supply to the injured brain using multimodal brain monitoring.

Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila.

Ionotropic glutamate receptors (iGluRs) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. We have characterized a family of iGluR-related genes in Drosophila, which we name ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead us to propose that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues.

Phase II study of imatinib in patients with recurrent gliomas of various histologies: a European Organisation for Research and Treatment of Cancer Brain Tumor Group Study.

PURPOSE: To evaluate the safety and the efficacy of imatinib in recurrent malignant gliomas. PATIENTS: AND METHODS: This was a single-arm, phase II study. Eligible patients had recurrent glioma after prior radiotherapy with an enhancing lesion on magnetic resonance imaging. Three different histologic groups were studied: glioblastomas (GBM), pure/mixed (anaplastic) oligodendrogliomas (OD), and low-grade or anaplastic astrocytomas (A). Imatinib was started at a dose of 600 mg/d with dose escalation to 800 mg in case of no toxicity; during the trial this dose was increased to 800 mg/d with escalation to 1,000 mg/d. Trial design was one-stage Fleming; both an objective response and 6 months of progression-free survival (PFS) were considered a successful outcome to treatment. RESULTS: A total of 112 patients (51 patients with GBM, 25 patients with A, and 36 patients with OD) were enrolled. Imatinib was in general well tolerated. The median number of cycles was 2.0 (range, 1 to 43 cycles). Five patients had an objective partial response, including three patients with GBM; all had 6 months of PFS. The 6-month PFS rate was 16% (95% CI, 8.0% to 34.0%) in GBM, 4.0% (95% CI, 0.3% to 15.0%) in OD, and 9% (95% CI, 2.0% to 25.0%) in A. The exposure to imatinib was significantly lower in patients using enzyme-inducing antiepileptic drugs. The presence of ABCG2 point mutations were not correlated with pharmacokinetic findings. No somatic activating mutations of KIT or platelet-derived growth factor receptor-A or -B were found. CONCLUSION: In the dose range of 600 to 1,000 mg/d, single-agent imatinib is well tolerated but has limited antitumor activity in patients with recurrent gliomas.

Molecular determinants of vesicular storage and release of the gliotransmitter D-serine from cortical astrocytes

Neuron-astrocyte reciprocal communication at synapses has emerged as a novel signalling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of 'gliotransmitters' such as glutamate, ATP or D-serine. A calcium-dependent exocytosis is proposed to drive the release of gliotransmitters but its existence is still debated. Over the last years, we have been studying the molecular determinants governing D-serine release from glia using different approaches. Using a novel bioassay for D-serine, we have been able to show that D-serine release occurs mainly through a calcium- and SNARE proteindependent mechanism just supporting the idea that this amino acid is released by exocytosis from glia. We next have pursued our exploration by confocal imaging and tracking of the exocytotic routes for Dserine- mediated gliotransmission and have shown that D-serine releasable pools are confined to synaptobrevin2/cellubrevin-bearing vesicles. To shed light onto the mechanisms controlling the storage and the release of gliotransmitters and namely D-serine, we have developed a new method for the immunoisolation of synaptobrevin 2- positive vesicles from rat cortical astrocytes in culture while preserving their content in gliotransmitters. The purified organelles are clear round shape vesicles of excellent purity with homogeneous size (40 nm) as judged by electron microscopy. Immunoblotting analysis revealed that isolated vesicles contain most of the major proteins already described for neuron-derived vesicles like synaptic vesicle protein 2 (SV2) and the proton pump H?-ATPase. In addition, we have analyzed the content for various amino acids of these vesicles by means of chiral capillary electrophoresis coupled to laser-induced fluorescence detection. The purified vesicles contain large amount of D-serine. We also detect peaks corresponding to unidentified compounds that may correspond to others amino acids. Postembedding immunogold labelling of the rat neocortex further revealed the expression of D-serine in astrocytes processes contacting excitatory synapses. Finally, we have examined the uptake properties for Dserine and glutamate inside the isolated glial vesicles. Our results provide significant support for the existence of an uptake system for D-serine in secretory glial vesicles and for the storage of chemical substances like D-serine and glutamate. 11th International Congress on Amino Acids, Peptides and Proteins 763 123

Doublecortin-positive cells in the adult primate cerebral cortex and possible role in brain plasticity and development.

We have demonstrated that cortical cell autografts might be a useful therapy in two monkey models of neurological disease: motor cortex lesion and Parkinson's disease. However, the origin of the useful transplanted cells obtained from cortical biopsies is not clear. In this report we describe the expression of doublecortin (DCX) in these cells based on reverse-transcription polymerase chain reaction (RT-PCR) and immunodetection in the adult primate cortex and cell cultures. The results showed that DCX-positive cells were present in the whole primate cerebral cortex and also expressed glial and/or neuronal markers such as glial fibrillary protein (GFAP) or neuronal nuclei (NeuN). We also demonstrated that only DCX/GFAP positive cells were able to proliferate and originate progenitor cells in vitro. We hypothesize that these DCX-positive cells in vivo have a role in cortical plasticity and brain reaction to injury. Moreover, in vitro these DCX-positive cells have the potential to reacquire progenitor characteristics that confirm their potential for brain repair.

The size and burden of mental disorders and other disorders of the brain in Europe 2010.

Aims: To provide 12-month prevalence and disability burden estimates of a broad range of mental and neurological disorders in the European Union (EU) and to compare these findings to previous estimates. Referring to our previous 2005 review, improved up-to-date data for the enlarged EU on a broader range of disorders than previously covered are needed for basic, clinical and public health research and policy decisions and to inform about the estimated number of persons affected in the EU. Method: Stepwise multi-method approach, consisting of systematic literature reviews, reanalyses of existing data sets, national surveys and expert consultations. Studies and data from all member states of the European Union (EU-27) plus Switzerland, Iceland and Norway were included. Supplementary information about neurological disorders is provided, although methodological constraints prohibited the derivation of overall prevalence estimates for mental and neurological disorders. Disease burden was measured by disability adjusted life years (DALY). Results: Prevalence: It is estimated that each year 38.2% of the EU population suffers from a mental disorder. Adjusted for age and comorbidity, this corresponds to 164.8 million persons affected. Compared to 2005 (27.4%) this higher estimate is entirely due to the inclusion of 14 new disorders also covering childhood/adolescence as well as the elderly. The estimated higher number of persons affected (2011: 165 m vs. 2005: 82 m) is due to coverage of childhood and old age populations, new disorders and of new EU membership states. The most frequent disorders are anxiety disorders (14.0%), insomnia (7.0%), major depression (6.9%), somatoform (6.3%), alcohol and drug dependence (>4%), ADHD (5%) in the young, and dementia (1-30%, depending on age). Except for substance use disorders and mental retardation, there were no substantial cultural or country variations. Although many sources, including national health insurance programs, reveal increases in sick leave, early retirement and treatment rates due to mental disorders, rates in the community have not increased with a few exceptions (i.e. dementia). There were also no consistent indications of improvements with regard to low treatment rates, delayed treatment provision and grossly inadequate treatment. Disability: Disorders of the brain and mental disorders in particular, contribute 26.6% of the total all cause burden, thus a greater proportion as compared to other regions of the world. The rank order of the most disabling diseases differs markedly by gender and age group; overall, the four most disabling single conditions were: depression, dementias, alcohol use disorders and stroke. Conclusion: In every year over a third of the total EU population suffers from mental disorders. The true size of "disorders of the brain" including neurological disorders is even considerably larger. Disorders of the brain are the largest contributor to the all cause morbidity burden as measured by DALY in the EU. No indications for increasing overall rates of mental disorders were found nor of improved care and treatment since 2005; less than one third of all cases receive any treatment, suggesting a considerable level of unmet needs. We conclude that the true size and burden of disorders of the brain in the EU was significantly underestimated in the past.Concerted priority action is needed at all levels, including substantially increased funding for basic, clinical and public health research in order to identify better strategies for improved prevention and treatment for isorders of the brain as the core health challenge of the 21st century. (C) 2011 Published by Elsevier B.V.

An in vivo ultrahigh field 14.1 T (1) H-MRS study on 6-OHDA and α-synuclein-based rat models of Parkinson's disease: GABA as an early disease marker.

The detection of Parkinson's disease (PD) in its preclinical stages prior to outright neurodegeneration is essential to the development of neuroprotective therapies and could reduce the number of misdiagnosed patients. However, early diagnosis is currently hampered by lack of reliable biomarkers. (1) H magnetic resonance spectroscopy (MRS) offers a noninvasive measure of brain metabolite levels that allows the identification of such potential biomarkers. This study aimed at using MRS on an ultrahigh field 14.1 T magnet to explore the striatal metabolic changes occurring in two different rat models of the disease. Rats lesioned by the injection of 6-hydroxydopamine (6-OHDA) in the medial-forebrain bundle were used to model a complete nigrostriatal lesion while a genetic model based on the nigral injection of an adeno-associated viral (AAV) vector coding for the human α-synuclein was used to model a progressive neurodegeneration and dopaminergic neuron dysfunction, thereby replicating conditions closer to early pathological stages of PD. MRS measurements in the striatum of the 6-OHDA rats revealed significant decreases in glutamate and N-acetyl-aspartate levels and a significant increase in GABA level in the ipsilateral hemisphere compared with the contralateral one, while the αSyn overexpressing rats showed a significant increase in the GABA striatal level only. Therefore, we conclude that MRS measurements of striatal GABA levels could allow for the detection of early nigrostriatal defects prior to outright neurodegeneration and, as such, offers great potential as a sensitive biomarker of presymptomatic PD.

Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress.

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.

Abundant Occurrence of Basal Radial Glia in the Subventricular Zone of Embryonic Neocortex of a Lissencephalic Primate, the Common Marmoset Callithrix jacchus.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Proton T1 relaxation times of metabolites in human occipital white and gray matter at 7 T.

Proton T1 relaxation times of metabolites in the human brain have not previously been published at 7 T. In this study, T1 values of CH3 and CH2 group of N-acetylaspartate and total creatine as well as nine other brain metabolites were measured in occipital white matter and gray matter at 7 T using an inversion-recovery technique combined with a newly implemented semi-adiabatic spin-echo full-intensity acquired localized spectroscopy sequence (echo time = 12 ms). The mean T1 values of metabolites in occipital white matter and gray matter ranged from 0.9 to 2.2 s. Among them, the T1 of glutathione, scyllo-inositol, taurine, phosphorylethanolamine, and N-acetylaspartylglutamate were determined for the first time in the human brain. Significant differences in T1 between white matter and gray matter were found for water (-28%), total choline (-14%), N-acetylaspartylglutamate (-29%), N-acetylaspartate (+4%), and glutamate (+8%). An increasing trend in T1 was observed when compared with previously reported values of N-acetylaspartate (CH3 ), total creatine (CH3 ), and total choline at 3 T. However, for N-acetylaspartate (CH3 ), total creatine, and total choline, no substantial differences compared to previously reported values at 9.4 T were discernible. The T1 values reported here will be useful for the quantification of metabolites and signal-to-noise optimization in human brain at 7 T. Magn Reson Med 69:931-936, 2013. © 2012 Wiley Periodicals, Inc.

Noise in Brain Activity Engenders Perception and Influences Discrimination Sensitivity.

Behavioral and brain responses to identical stimuli can vary with experimental and task parameters, including the context of stimulus presentation or attention. More surprisingly, computational models suggest that noise-related random fluctuations in brain responses to stimuli would alone be sufficient to engender perceptual differences between physically identical stimuli. In two experiments combining psychophysics and EEG in healthy humans, we investigated brain mechanisms whereby identical stimuli are (erroneously) perceived as different (higher vs lower in pitch or longer vs shorter in duration) in the absence of any change in the experimental context. Even though, as expected, participants' percepts to identical stimuli varied randomly, a classification algorithm based on a mixture of Gaussians model (GMM) showed that there was sufficient information in single-trial EEG to reliably predict participants' judgments of the stimulus dimension. By contrasting electrical neuroimaging analyses of auditory evoked potentials (AEPs) to the identical stimuli as a function of participants' percepts, we identified the precise timing and neural correlates (strength vs topographic modulations) as well as intracranial sources of these erroneous perceptions. In both experiments, AEP differences first occurred ∼100 ms after stimulus onset and were the result of topographic modulations following from changes in the configuration of active brain networks. Source estimations localized the origin of variations in perceived pitch of identical stimuli within right temporal and left frontal areas and of variations in perceived duration within right temporoparietal areas. We discuss our results in terms of providing neurophysiologic evidence for the contribution of random fluctuations in brain activity to conscious perception.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.