Monitoring regional astrocyte diversity by cell type-specific proteomic labeling in vivo.

Astrocytes exhibit regional heterogeneity in morphology, function and molecular composition to support and modulate neuronal function and signaling in a region-specific manner. To characterize regional heterogeneity of astrocytic proteomes of different brain regions we established an inducible Aldh1l1-methionyl-tRNA-synthetaseL274G (MetRSL274G ) mouse line that allows astrocyte-specific metabolic labeling of newly synthesized proteins by azidonorleucine (ANL) in vivo and subsequent isolation of tagged proteins by click chemistry. We analyzed astrocytic proteins from four different brain regions by mass spectrometry. The induced expression of MetRSL274G is restricted to astrocytes and identified proteins show a high overlap with proteins compiled in "AstroProt," a newly established database for astrocytic proteins. Gene enrichment analysis reveals a high similarity among brain regions with subtle differences in enriched biological processes and in abundances of key astrocytic proteins for hippocampus, cortex and striatum. However, the cerebellar proteome stands out with proteins being highly associated with the calcium signaling pathway or with bipolar disorder. Subregional analysis of single astrocyte TAMRA intensities in hippocampal layers indicates distinct subregional heterogeneity of astrocytes and highlights the applicability of our toolbox to study differences of astrocytic proteomes in vivo.

A role for TASK2 channels in the human immunological synapse.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

14-3-3 Proteins regulate K2P 5.1 surface expression on T lymphocytes.

K2P 5.1 channels (also called TASK-2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K2P 5.1 channels in vitro provokes enhanced T-cell effector functions. However, the molecular mechanisms regulating intracellular K2P 5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K2P 5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14-3-3 proteins as novel binding partners of K2P 5.1 channels. We show that a non-classical 14-3-3 consensus motif (R-X-X-pT/S-x) at the channel's C-terminus allows the binding between K2P 5.1 and 14-3-3. The mutant K2P 5.1/S266A diminishes the protein-protein interaction and reduces the amplitude of membrane currents. Application of a non-peptidic 14-3-3 inhibitor (BV02) significantly reduces the number of wild-type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T-cell effector functions. Taken together, we demonstrate that 14-3-3 interacts with K2P 5.1 and plays an important role in channel trafficking.

Deficiency of the immunoproteasome subunit ß5i/LMP7 supports the anxiogenic effects of mild stress and facilitates cued fear memory in mice.

Proteolysis as mediated by one of the major cellular protein degradation pathways, the ubiquitin-proteasome system (UPS), plays an essential role in learning and memory formation. However, the functional relevance of immunoproteasomes in the healthy brain and especially their impact on normal brain function including processes of learning and memory has not been investigated so far. In the present study, we analyzed the phenotypic effects of an impaired immunoproteasome formation using a ß5i/LMP7-deficient mouse model in different behavioral paradigms focusing on locomotor activity, exploratory behavior, innate anxiety, startle response, prepulse inhibition, as well as fear and safety conditioning. Overall, our results demonstrate no strong effects of constitutive ß5i/LMP7-deficiency on gross locomotor abilities and anxiety-related behavior in general. However, ß5i/LMP7-deficient mice expressed more anxiety after mild stress and increased cued fear after fear conditioning. These findings indicate that the basal proper formation of immunoproteasomes and/or at least the expression of ß5i/LMP7 in healthy mice seem to be involved in the regulation of anxiety and cued fear levels.

Sumoylation in astrocytes induces changes in the proteome of the derived small extracellular vesicles which change protein synthesis and dendrite morphology in target neurons.

Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.

Deoxynivalenol affects cell metabolism in vivo and inhibits protein synthesis in IPEC-1 cells.

Deoxynivalenol is present in forage crops in concentrations that endanger animal welfare but is also found in cereal-based food. The amphipathic nature of mycotoxins allows them to cross the cell membrane and interacts with different cell organelles such as mitochondria and ribosomes. In our study, we investigated the gene expression of several genes in vivo and in vitro that are related to the metabolism. We observed a significantly higher COX5B and MHCII expression in enterocytes of DON-fed pigs compared to CON-fed pigs and a marked increase in GAPDH and SLC7A11 in DON-fed pigs, but we could not confirm this in vitro in IPEC-1. In vitro, functional metabolic analyses were performed with a seahorse analyzer. A significant increase of non-mitochondrial respiration was observed in all DON-treatment groups (50-2000 ng/mL). The oxygen consumption of cells, which were cultured on membranes, was examined with a fiber-glass electrode. Here, we found significantly lower values for DON 200- and DON 2000-treatment group. The effect on ribosomes was investigated using biorthogonal non-canonical amino acid tagging (BONCAT) to tag newly synthesized proteins. A significantly reduced amount was found in almost all DON-treatment groups. Our findings clearly show that apical and basolateral DON-treatment of epithelial cell layer results in decreasing amounts of newly synthesized proteins. Furthermore, our study shows that DON affects enterocyte metabolism in vivo and in vitro.

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

The survival promoting peptide Y-P30 promotes cellular migration.

Y-P30, the 30 amino acid N-terminal peptide of the dermcidin gene, has been found to promote neuronal survival and differentiation. Its early presence in development and import to the fetal brain led to the hypothesis that Y-P30 has an influence on proliferation, differentiation and migration. Neurospheres derived from neural stem cells isolated from E13 mouse cortex and striatal ganglionic eminences were treated with Y-P30, however, the proportion of progenitors, neurons and astrocytes generated in differentiation assays was not altered. A short Y-P30 treatment of undifferentiated striatal and cortical neurospheres failed to alter the proportion of BrdU-positive cells. A longer treatment reduced the percentage of BrdU-positive cells and GABA-immunoreactive neurons only in striatal spheres. The presence of Y-P30 enhanced migration of T24 human bladder carcinoma cells in a wound-healing assay in vitro. Further, Y-P30 enhanced migration of T24 cells, rat primary cortical astrocytes and PC12 cells in chemotactic Boyden chamber assays. Together, these findings suggest that a major function of Y-P30 is to promote migration of neural and non-neural cell types.

Calneurons provide a calcium threshold for trans-Golgi network to plasma membrane trafficking.

Phosphatidylinositol 4-OH kinase IIIbeta (PI-4Kbeta) is involved in the regulated local synthesis of phospholipids that are crucial for trans-Golgi network (TGN)-to-plasma membrane trafficking. In this study, we show that the calcium sensor proteins calneuron-1 and calneuron-2 physically associate with PI-4Kbeta, inhibit the enzyme profoundly at resting and low calcium levels, and negatively interfere with Golgi-to-plasma membrane trafficking. At high calcium levels this inhibition is released and PI-4Kbeta is activated via a preferential association with neuronal calcium sensor-1 (NCS-1). In accord to its supposed function as a filter for subthreshold Golgi calcium transients, neuronal overexpression of calneuron-1 enlarges the size of the TGN caused by a build-up of vesicle proteins and reduces the number of axonal Piccolo-Bassoon transport vesicles, large dense core vesicles that carry a set of essential proteins for the formation of the presynaptic active zone during development. A corresponding protein knockdown has the opposite effect. The opposing roles of calneurons and NCS-1 provide a molecular switch to decode local calcium transients at the Golgi and impose a calcium threshold for PI-4Kbeta activity and vesicle trafficking.

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.

Regulation of CREB Phosphorylation in Nucleus Accumbens after Relief Conditioning.

Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKIIα, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning.

Cytotoxic CD8+ T cell-neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death.

Cytotoxic CD8(+) T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8(+) T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8(+) T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca(2+) levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within approximately 1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8(+) T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.

Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition.

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.

Intracellular Ca2+ release-dependent inactivation of Ca2+ currents in thalamocortical relay neurons.

Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.

Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila.

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

CNS inflammation and neuronal degeneration is aggravated by impaired CD200-CD200R-mediated macrophage silencing.

Multiple sclerosis is a chronic disabling CNS disorder, characterized by autoimmune inflammatory demyelination and neurodegeneration. CD200, broadly expressed on neurons and endothelial cells, mediates inhibitory signals through its receptor, CD200R, on cells of myeloid origin. Antibody-mediated blockade of CD200R leads to an aggravated clinical course of rodent experimental autoimmune encephalomyelitis in vivo, accompanied by profoundly augmented cellular infiltrates consisting of T cells and activated iNOS(+) macrophages in inflammatory spinal cord lesions. In vitro blockade of CD200R on macrophages leads to enhanced IFN-gamma-induced release of IL6 and neuronal cell death in co-cultures with hippocampal neurons expressing CD200. CD200 and its receptor could also be detected on neurons and macrophages in human MS plaques. Therefore the CD200-CD200R pathway seems of critical relevance for macrophage-mediated damage in autoimmune inflammation of the CNS.

Deoxynivalenol Affects Cell Metabolism and Increases Protein Biosynthesis in Intestinal Porcine Epithelial Cells (IPEC-J2): DON Increases Protein Biosynthesis.

Deoxynivalenol (DON) is a toxin found in cereals as well as in processed products such as pasta, and causes substantial economic losses for stock breeding as it induces vomiting, reduced feeding, and reduced growth rates in piglets. Oxidative phosphorylation, TCA-cycle, transcription, and translation have been hypothesized to be leading pathways that are affected by DON. We used an application of high and low glucose to examine oxidative phosphorylation and anaerobic glycolysis. A change in the metabolic status of IPEC-J2 was observed and confirmed by microarray data. Measurements of oxygen consumption resulted in a significant reduction, if DON attacks from the basolateral. Furthermore, we found a dose-dependent effect with a significant reduction at 2000 ng/mL. In addition, SLC7A11 and PHB, the genes with the highest regulation in our microarray analyses under low glucose supply, were investigated and showed a variable regulation on protein level. Lactate production and glucose consumption was investigated to examine the impact of DON on anaerobic glycolysis and we observed a significant increase in 2000 blhigh and a decrease in 2000 aphigh. Interestingly, both groups as well as 200 blhigh showed a significant higher de novo protein synthesis when compared to the control. These results indicate the direct or indirect impact of DON on metabolic pathways in IPEC-J2.

Low Neurotoxicity of ONX-0914 Supports the Idea of Specific Immunoproteasome Inhibition as a Side-Effect-Limiting, Therapeutic Strategy.

Application of the proteasome inhibitor Bortezomib for the treatment of haematopoietic malignancies such as multiple myeloma significantly improves the average overall survival of patients. However, one of the most severe side effects is the development of peripheral neuropathies caused by neurotoxic effects of Bortezomib limiting its therapeutic efficacy. With ONX-0914 a specific inhibitor of the ß5i (LMP7)-immunosubunit containing proteasomes was developed that targets exclusively the proteasome subtypes mainly expressed in immune cells including B lymphocytes as the origin of multiple myeloma. Furthermore, immunosubunitspecific inhibitors have been shown to be promising tools for the therapy of autoimmune disorders. In the presented study, we analysed the concentration-dependent impact of both inhibitors on primary neurons regarding survival rate, morphological changes, and overall viability. Our results clearly demonstrate that ONX-0914, compared to Bortezomib, is less neurotoxic suggesting its potential as a putative antineoplastic drug and as a candidate for the treatment of autoimmune disorders affecting the peripheral and/or central nervous system.

A maternal blood-borne factor promotes survival of the developing thalamus.

In this report, we describe the identification of a polypeptide survival-promoting factor that is produced by maternal and early postnatal peripheral blood mononuclear cells (PBMCs) of the immune system in Long-Evans rats and humans. The factor, termed Y-P30, most likely arises from proteolytic processing of a larger precursor protein and accumulates mainly in pyramidal neurons of the developing cortex and hippocampus but not in astrocytes. It was released from neurons grown in culture and substantially promotes survival of cells in explant monocultures of perinatal thalamus from the offspring. Y-P30 mRNA was not detectable in infant or adult brain and was present only in blood cells of pregnant rats and humans but not in nonpregnant controls. However, Y-P30 transcription could be induced in PBMCs of adult animals by a central nervous system lesion (i.e., optic nerve crush), which points to a potential role of the factor not only in neuronal development but also in neuroinflammation after white matter injury.