Paroxysmal dystonia results from the loss of RIM4 in Purkinje cells.

Full-length RIM1 and 2 are key components of the presynaptic active zone that ubiquitously control excitatory and inhibitory neurotransmitter release. Here, we report that the function of the small RIM isoform RIM4, consisting of a single C2 domain, is strikingly different from that of the long isoforms. RIM4 is dispensable for neurotransmitter release but plays a postsynaptic, cell-type specific role in cerebellar Purkinje cells that is essential for normal motor function. In the absence of RIM4, Purkinje cell intrinsic firing is reduced and caffeine-sensitive, and dendritic integration of climbing fibre input is disturbed. Mice lacking RIM4, but not mice lacking RIM1/2, selectively in Purkinje cells exhibit a severe, hours-long paroxysmal dystonia. These episodes can also be induced by caffeine, ethanol or stress and closely resemble the deficits seen with mutations of the PNKD (paroxysmal non-kinesigenic dystonia) gene. Our data reveal essential postsynaptic functions of RIM proteins and show non-overlapping specialized functions of a small isoform despite high homology to a single domain in the full-length proteins.

Ganglioglioma cells potentiate neuronal network synchronicity and elicit burst discharges via released factors.

Gangliogliomas (GGs) represent the most frequent glioneuronal tumor entity associated with chronic recurrent seizures; rare anaplastic GGs variants retain the glioneuronal character. So far, key mechanisms triggering chronic hyperexcitability in the peritumoral area are unresolved. Based on a recent mouse model for anaplastic GG (BRAFV600E, mTOR activation and Trp53KO) we here assessed the influence of GG-secreted factors on non-neoplastic cells in-vitro. We generated conditioned medium (CM) from primary GG cell cultures to developing primary cortical neurons cultured on multielectrode-arrays and assessed their electrical activity in comparison to neurons incubated with naïve and neuronal CMs. Our results showed that the GG CM, while not affecting the mean firing rates of networks, strongly accelerated the formation of functional networks as indicated increased synchrony of firing and burst activity. Washing out the GG CM did not reverse these effects indicating an irreversible effect on the neuronal network. Mass spectrometry analysis of GG CM detected several enriched proteins associated with neurogenesis as well as gliogenesis, including Gap43, App, Apoe, S100a8, Tnc and Sod1. Concomitantly, immunocytochemical analysis of the neuronal cultures exposed to GG CM revealed abundant astrocytes suggesting that the GG-secreted factors induce astroglial proliferation. Pharmacological inhibition of astrocyte proliferation only partially reversed the accelerated network maturation in neuronal cultures exposed to GG CM indicating that the GG CM exerts a direct effect on the neuronal component. Taken together, we demonstrate that GG-derived paracrine signaling alone is sufficient to induce accelerated neuronal network development accompanied by astrocytic proliferation. Perspectively, a deeper understanding of factors involved may serve as the basis for future therapeutic approaches.

Early cortical oligodendrocyte precursor cells are transcriptionally distinct and lack synaptic connections.

Oligodendrocyte precursor cells (OPCs) generate oligodendrocytes, a process that may be tuned by neuronal activity, possibly via synaptic connections to OPCs. However, a developmental role of synaptic signaling to OPCs has so far not been shown unequivocally. To address this question, we comparatively analyzed functional and molecular characteristics of highly proliferative and migratory OPCs in the embryonic brain. Embryonic OPCs in mice (E18.5) shared the expression of voltage-gated ion channels and their dendritic morphology with postnatal OPCs, but almost completely lacked functional synaptic currents. Transcriptomic profiling of PDGFRα+ OPCs revealed a limited abundance of genes coding for postsynaptic signaling and synaptogenic cell adhesion molecules in the embryonic versus the postnatal period. RNA sequencing of single OPCs showed that embryonic synapse-lacking OPCs are found in clusters distinct from postnatal OPCs and with similarities to early progenitors. Furthermore, single-cell transcriptomics demonstrated that synaptic genes are transiently expressed only by postnatal OPCs until they start to differentiate. Taken together, our results indicate that embryonic OPCs represent a unique developmental stage biologically resembling postnatal OPCs but without synaptic input and a transcriptional signature in the continuum between OPCs and neural precursors.

Ste20-like Kinase Is Critical for Inhibitory Synapse Maintenance and Its Deficiency Confers a Developmental Dendritopathy.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Publisher Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Author Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation.

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Drebrin Autoantibodies in Patients with Seizures and Suspected Encephalitis.

OBJECTIVE: Assess occurrence of the dendritic spine scaffolding protein Drebrin as a pathophysiologically relevant autoantibody target in patients with recurrent seizures and suspected encephalitis as leading symptoms. METHODS: Sera of 4 patients with adult onset epilepsy and suspected encephalitis of unresolved etiology and equivalent results in autoantibody screening were subjected to epitope identification. We combined a wide array of approaches, ranging from immunoblotting, immunoprecipitation, mass spectrometry, subcellular binding pattern analyses in primary neuronal cultures, and immunohistochemistry in brains of wild-type and Drebrin knockout mice to in vitro analyses of impaired synapse formation, morphology, and aberrant neuronal excitability by antibody exposure. RESULTS: In the serum of a patient with adult onset epilepsy and suspected encephalitis, a strong signal at ∼70kDa was detected by immunoblotting, for which mass spectrometry revealed Drebrin as the putative antigen. Three other patients whose sera also showed strong immunoreactivity around 70kDa on Western blotting were also anti-Drebrin-positive. Seizures, memory impairment, and increased protein content in cerebrospinal fluid occurred in anti-Drebrin-seropositive patients. Alterations in cerebral magnetic resonance imaging comprised amygdalohippocampal T2-signal increase and hippocampal sclerosis. Diagnostic biopsy revealed T-lymphocytic encephalitis in an anti-Drebrin-seropositive patient. Exposure of primary hippocampal neurons to anti-Drebrin autoantibodies resulted in aberrant synapse composition and Drebrin distribution as well as increased spike rates and the emergence of burst discharges reflecting network hyperexcitability. INTERPRETATION: Anti-Drebrin autoantibodies define a chronic syndrome of recurrent seizures and neuropsychiatric impairment as well as inflammation of limbic and occasionally cortical structures. Immunosuppressant therapies should be considered in this disorder. ANN NEUROL 2020;87:869-884.

Calcium Channel Subunit α2δ4 Is Regulated by Early Growth Response 1 and Facilitates Epileptogenesis.

Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca2+ channel (VDCC) subunit CaV3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits CaV3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced "transcriptional Ca2+ channelopathy."SIGNIFICANCE STATEMENT The onset of focal recurrent seizures often occurs after an epileptogenic process induced by transient insults to the brain. Recently, transcriptional control mechanisms for individual genes involved in converting neurons hyperexcitable have been identified, including early growth response 1 (Egr1), which activates transcription of the T-type Ca2+ channel subunit CaV3.2. Here, we find Egr1 to regulate also the expression of the voltage-dependent Ca2+ channel subunit α2δ4, which was augmented after pilocarpine- and kainic acid-induced status epilepticus. In addition, we observed that α2δ4 affected spontaneous network activity and the susceptibility for seizure induction. Furthermore, we detected corresponding dynamics in human biopsies from epilepsy patients. In conclusion, Egr1 orchestrates a seizure-induced "transcriptional Ca2+ channelopathy" consisting of CaV3.2 and α2δ4, which act synergistically in epileptogenesis.

Systemic High-Mobility Group Box-1: A Novel Predictive Biomarker for Cerebral Vasospasm in Aneurysmal Subarachnoid Hemorrhage.

OBJECTIVES: To investigate the release of proinflammatory damage-associated molecular pattern molecule "high-mobility group box-1" in the serum of patients after aneurysmal subarachnoid hemorrhage and its association with cerebral vasospasm. DESIGN: Retrospective observational study. SETTING: University hospital. PATIENTS: Aneurysmal subarachnoid hemorrhage patients admitted within 24 hours of ictus. INTERVENTIONS: Standard subarachnoid hemorrhage treatment after clipping or coiling of aneurysm. MEASUREMENTS AND MAIN RESULTS: We enrolled 53 aneurysmal subarachnoid hemorrhage patients from which peripheral venous blood was withdrawn on days 1, 3, 5, 7, 9, 11, and 13 and once from the controls to obtain the serum. Serum high-mobility group box-1 concentration was quantified by enzyme-linked immunosorbent assay. Serum interleukin-6 and peripheral blood leukocytes were also determined over the first 2 weeks after subarachnoid hemorrhage. Patients' data were recorded prospectively. Serum high-mobility group box-1 was significantly elevated in subarachnoid hemorrhage patients from day 1 to day 13 when compared with nonsubarachnoid hemorrhage patients (p < 0.05). Patients with cerebral vasospasm showed significantly higher high-mobility group box-1 starting from day 1 to day 13 when compared with patients without cerebral vasospasm. Cumulative levels of high-mobility group box-1 showed significant correlation with peripheral blood leukocytes and interleukin-6 levels (p < 0.05). Receiver operating characteristic curve analysis showed that serum high-mobility group box-1 level at admission may be a predictive biomarker for cerebral vasospasm with a sensitivity of 59% and a specificity of 82% at a cutoff value of 5.6 ng/mL. CONCLUSIONS: Serum high-mobility group box-1 is differentially elevated after subarachnoid hemorrhage. Serum high-mobility group box-1 levels were elevated early after subarachnoid hemorrhage (day 1) and remained significantly high until day 13 in patients who developed cerebral vasospasm. Our data suggest that serum high-mobility group box-1 may be a predictive biomarker for the detection of CVS.

Transient Cnp expression by early progenitors causes Cre-Lox-based reporter lines to map profoundly different fates.

NG2 expressing oligodendroglial precursor cells are ubiquitous in the central nervous system and the only cell type cycling throughout life. Previous fate mapping studies have remained inconsistent regarding the question whether NG2 cells are capable of generating certain types of neurons. Here, we use CNP-Cre mice to map the fate of a sub-population of NG2 cells assumed to be close to differentiation. When crossing these mice with the ROSA26/YFP Cre-reporter line we discovered large numbers of reporter-expressing pyramidal neurons in the piriform and dorsal cortex. In contrast, when using Z/EG reporter mice to track the fate of Cnp-expressing NG2 cells only oligodendroglial cells were found reporter positive. Using BrdU-based birth dating protocols and inducible NG2CreER:ROSA26/YFP mice we show that YFP positive neurons are generated from radial glial cells and that these radial glial cells display temporary and low level activity of certain oligodendroglial genes sufficient to recombine the Cre-inducible reporter gene in ROSA26/YFP but not in Z/EG mice. Taken together, we did not obtain evidence for generation of neurons from NG2 cells. Our results suggest that with an appropriate reporter system Cnp activity can be used to define a proliferative subpopulation of NG2 cells committed to generate oligodendrocytes. However, the strikingly different results obtained from ROSA26/YFP versus Z/EG mice demonstrate that the choice of Cre-reporter line can be of crucial importance for fate mapping studies and other applications of the Cre-lox technology. GLIA 2017;65:342-359.

Aneurysmal subarachnoid hemorrhage lead to systemic upregulation of IL-23/IL-17 inflammatory axis.

IL-23 and IL-17 are pro-inflammatory cytokines. IL-23 is secreted by activated macrophages and dendritic cells, while IL-17 by Th17 cells. Serum IL-23 and IL-17 are known to be elevated in numerous inflammatory diseases including neurodegenerative diseases. The role of serum IL-23 and IL-17 in aneurysmal subarachnoid hemorrhage (aSAH) has still not been investigated. The present work investigates the serum IL-23 and IL-17 levels and their association with post hemorrhagic complications and clinical outcome in patients with aSAH. METHODS: In this study, 80 patients with aSAH (Hunt and Hess grade I-V) were prospectively recruited. We enrolled 24 control patients with lumbar spinal stenosis. Peripheral venous blood was withdrawn from controls and from aSAH patients at day 1 and day 7, allowed to clot and centrifuged to obtain serum. Enzyme linked immunoassay kits were employed to quantify the serum levels of IL-23 and IL-17 by applying 50µL of serum samples. Post hemorrhagic complications and clinical outcome were documented prospectively from patient's hospital record. RESULTS: Serum IL-23 and IL-17 levels were significantly elevated in aSAH patients at day 1 and day 7 (n=80) as compared to control patients (n=24). Further analysis after dichotomy of patients who suffered from post hemorrhagic complications including cerebral vasospasm, chronic hydrocephalus, seizures, cerebral ischemia, delayed neurological deficits showed differential correlations with different post hemorrhagic complications (Table 1). Serum IL-23 and IL-17 levels did not correlate with clinical outcome. CONCLUSION: Serum IL-23 and IL-17 levels were elevated in patients with aSAH showing upregulation of IL-23/IL-17 inflammatory axis after aSAH. Serum IL-23 and IL-17 showed differential correlations with post hemorrhagic complications and no correlation with clinical outcome.

Elevated Systemic IL-6 Levels in Patients with Aneurysmal Subarachnoid Hemorrhage Is an Unspecific Marker for Post-SAH Complications.

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is still a fatal and morbid disease, although bleeding aneurysms can be secured in almost all cases. Occurrence of post-SAH complications including cerebral vasospasm, delayed cerebral ischemia, hydrocephalus, epilepsy, and infections are the main determinants of clinical outcome. Hence, it is important to search for early predictors for specific post-SAH complications to treat these complications properly. Both cellular and molecular (cytokines) inflammation play a key role after aSAH during the phase of occurrence of post-SAH complications. Interleukin-6 (IL-6) is a well-known cytokine that has been extensively analyzed in cerebrospinal fluid (CSF) of patients after aSAH, but detailed studies exploring the role of systemic IL-6 in aSAH associated complications and its impact on early clinical outcome prediction are lacking. The current study aims to analyze the systemic IL-6 levels over two weeks after bleeding and its role in post-SAH complications. Methods: We recruited 80 aSAH patients prospectively who underwent peripheral venous blood withdrawal in serum gel tubes. The blood was centrifuged to harvest the serum, which was immediately frozen at -80 °C until analysis. Serum IL-6 levels were quantified using Immulite immunoassay system. Patient records including age, gender, post-SAH complications, aneurysm treatment, and clinical outcome (modified Rankin scale and Glasgow outcome scale) were retrieved to allow different subgroup analysis. Results: Serum IL-6 levels were significantly raised after aSAH compared to healthy controls over the first two weeks after hemorrhage. Serum IL-6 levels were found to be significantly elevated in aSAH patients presenting with higher Hunt and Hess grades, increasing age, and both intraventricular and intracerebral hemorrhage. Interestingly, serum IL-6 was also significantly raised in aSAH patients who developed seizures, cerebral vasospasm (CVS), and chronic hydrocephalus. IL-6 levels were sensitive to the development of infections and showed an increase in patients who developed pneumoniae. Intriguingly, we found a delayed increase in serum IL-6 in patients developing cerebral infarction. Finally, IL-6 levels were significantly higher in patients presenting with poor clinical outcome in comparison to good clinical outcome at discharge from hospital. Conclusion: Serum IL-6 levels were elevated early after aSAH and remained high over the two weeks after initial bleeding. Serum IL-6 was elevated in different aSAH associated complications, acting as a non-specific marker for post-SAH complications and an important biomarker for clinical outcome at discharge.

Tuning local calcium availability: cell-type-specific immobile calcium buffer capacity in hippocampal neurons.

It has remained difficult to ascribe a specific functional role to immobile or fixed intracellular calcium buffers in central neurons because the amount of these buffers is unknown. Here, we explicitly isolated the fixed buffer fraction by prolonged whole-cell patch-clamp dialysis and quantified its buffering capacity in murine hippocampal slices using confocal calcium imaging and the "added-buffer" approach. In dentate granule cells, the calcium binding ratio (κ) after complete washout of calbindin D28k (Cb), κfixed, displayed a substantial value of ∼100. In contrast, in CA1 oriens lacunosum moleculare (OLM) interneurons, which do not contain any known calcium-binding protein(s), κfixed amounted to only ∼30. Based on these values, a theoretical analysis of dendritic spread of calcium after local entry showed that fixed buffers, in the absence of mobile species, decrease intracellular calcium mobility 100- and 30-fold in granule cells and OLM cells, respectively, and thereby strongly slow calcium signals. Although the large κfixed alone strongly delays the spread of calcium in granule cells, this value optimizes the benefits of additionally expressing the mobile calcium binding protein Cb. With such high κfixed, Cb effectively increases the propagation velocity to levels seen in OLM cells and, contrary to expectation, does not affect the peak calcium concentration close to the source but sharpens the spatial and temporal calcium gradients. The data suggest that the amount of fixed buffers determines the temporal availability of calcium for calcium-binding partners and plays a pivotal role in setting the repertoire of cellular calcium signaling regimens.

RIM3γ and RIM4γ are key regulators of neuronal arborization.

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/ß and RIM2α/ß, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures and in vivo resulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.

Age-dependent fate and lineage restriction of single NG2 cells.

NG2-expressing glia (NG2 cells, polydendrocytes) appear in the embryonic brain, expand perinatally, and persist widely throughout the gray and white matter of the mature central nervous system. We have previously reported that NG2 cells generate oligodendrocytes in both gray and white matter and a subset of protoplasmic astrocytes in the gray matter of the ventral forebrain and spinal cord. To investigate the temporal changes in NG2 cell fate, we generated NG2creER™BAC transgenic mice, in which tamoxifen-inducible Cre is expressed in NG2 cells. Cre induction at embryonic day 16.5, postnatal day (P) 2, P30 and P60 in mice that were double transgenic for NG2creER™BAC and the Cre reporter revealed that NG2 cells in the postnatal brain generate only NG2 cells or oligodendrocytes, whereas NG2 cells in the embryonic brain generate protoplasmic astrocytes in the gray matter of the ventral forebrain in addition to oligodendrocytes and NG2 cells. Analysis of cell clusters from single NG2 cells revealed that more than 80% of the NG2 cells in the P2 brain give rise to clusters consisting exclusively of oligodendrocytes, whereas the majority of the NG2 cells in the P60 brain generate clusters that contain only NG2 cells or a mixture of oligodendrocytes and NG2 cells. Furthermore, live cell imaging of single NG2 cells from early postnatal brain slices revealed that NG2 cells initially divide symmetrically to produce two daughter NG2 cells and that differentiation into oligodendrocytes occurred after 2-3 days.

Multiomics of early epileptogenesis in mice reveals phosphorylation and dephosphorylation-directed growth and synaptic weakening.

To investigate the phosphorylation-based signaling and protein changes occurring early in epileptogenesis, the hippocampi of mice treated with pilocarpine were examined by quantitative mass spectrometry at 4 and 24 h post-status epilepticus at vast depth. Hundreds of posttranscriptional regulatory proteins were the major early targets of increased phosphorylation. At 24 h, many protein level changes were detected and the phosphoproteome continued to be perturbed. The major targets of decreased phosphorylation at 4 and 24 h were a subset of postsynaptic density scaffold proteins, ion channels, and neurotransmitter receptors. Many proteins targeted by dephosphorylation at 4 h also had decreased protein abundance at 24 h, indicating a phosphatase-mediated weakening of synapses. Increased translation was indicated by protein changes at 24 h. These observations, and many additional indicators within this multiomic resource, suggest that early epileptogenesis is characterized by signaling that stimulates both growth and a homeostatic response that weakens excitability.

Depression of release by mGluR8 alters Ca2+ dependence of release machinery.

The ubiquitous presynaptic metabotropic glutamate receptors (mGluRs) are generally believed to primarily inhibit synaptic transmission through blockade of Ca(2+) entry. Here, we analyzed how mGluR8 achieves a nearly complete inhibition of glutamate release at hippocampal synapses. Surprisingly, presynaptic Ca(2+) imaging and miniature excitatory postsynaptic current recordings showed that mGluR8 acts without affecting Ca(2+) entry, diffusion, and buffering. We quantitatively compared the Ca(2+) dependence of the inhibition of release by mGluR8 with the inhibition by ω-conotoxin GVIA. These calculations suggest that the inhibition produced by mGluR8 may be explained by a decrease in the apparent Ca(2+) affinity of the release sensor and, to a smaller extent, by a reduction of the maximal release rate. Upon activation of mGluR8, phasic transmitter release toward the end of a train of action potentials is greater as compared with presynaptic inhibition induced by blocking Ca(2+) entry, which is consistent with the important role of Ca(2+) in accelerating the replenishment of released vesicles. The action of mGluR8 was resistant to blockers of classical G-protein transduction pathways including inhibition of adenylate cyclase and may represent a direct effect on the release machinery. In conclusion, our data identify a mode of presynaptic inhibition which allows mGluR8 to profoundly inhibit vesicle fusion while not diminishing vesicle replenishment and which thereby differentially changes the temporal transmission properties of the inhibited synapse.

Local perfusion of capillaries reveals disrupted beta-amyloid homeostasis at the blood-brain barrier in Tg2576 murine Alzheimer's model.

BACKGROUND: Parenchymal accumulation of beta-amyloid (Aß) characterizes Alzheimer's disease (AD). Aß homeostasis is maintained by two ATP-binding cassette (ABC) transporters (ABCC1 and ABCB1) mediating efflux, and the receptor for advanced glycation end products (RAGE) mediating influx across the blood-brain barrier (BBB). Altered transporter levels and disruption of tight junctions (TJ) were linked to AD. However, Aß transport and the activity of ABCC1, ABCB1 and RAGE as well as the functionality of TJ in AD are unclear. METHODS: ISMICAP, a BBB model involving microperfusion of capillaries, was used to assess BBB properties in acute cortical brain slices from Tg2576 mice compared to wild-type (WT) controls using two-photon microscopy. TJ integrity was tested by vascularly perfusing biocytin-tetramethylrhodamine (TMR) and quantifying its extravascular diffusion as well as the diffusion of FM1-43 from luminal to abluminal membranes of endothelial cells (ECs). To assess ABCC1 and ABCB1 activity, calcein-AM was perfused, which is converted to fluorescent calcein in ECs and gets actively extruded by both transporters. To probe which transporter is involved, probenecid or Elacridar were applied, individually or combined, to block ABCC1 and ABCB1, respectively. To assess RAGE activity, the binding of 5-FAM-tagged Aß by ECs was quantified with or without applying FPS-ZM1, a RAGE antagonist. RESULTS: In Tg2576 mouse brain, extravascular TMR was 1.8-fold that in WT mice, indicating increased paracellular leakage. FM1-43 staining of abluminal membranes in Tg2576 capillaries was 1.7-fold that in WT mice, indicating reduced TJ integrity in AD. While calcein was undetectable in WT mice, its accumulation was significant in Tg2576 mice, suggesting lower calcein extrusion in AD. Incubation with probenecid or Elacridar in WT mice resulted in a marked calcein accumulation, yet probenecid alone had no effect in Tg2576 mice, implying the absence of probenecid-sensitive ABC transporters. In WT mice, Aß accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aß fluorescence was observed in Tg2576 vessels, and FPS-ZM1 was without effect, suggesting reduced RAGE binding activity. CONCLUSIONS: Disrupted TJ integrity, reduced ABCC1 functionality and decreased RAGE binding were identified as BBB alterations in Tg2576 mice, with the latter finding challenging the current concepts. Our results suggest to manage AD by including modulation of TJ proteins and Aß-RAGE binding.