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Abstract Background Parenchymal accumulation of beta-amyloid (Aβ) characterizes Alzheimer’s disease (AD). Aβ 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, Aβ 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 Aβ 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, Aβ accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aβ 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 Aβ-RAGE binding.

Summary: 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.

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.

The blood-brain barrier (BBB) tightly and dynamically regulates exchange with the brain. Here, the authors report a brain slice approach to study the native BBB in a highly controlled manner by combining intravascular perfusion and multiphoton microscopy.

Summary: Synaptically released glutamate is largely cleared by glutamate transporters localized on perisynaptic astrocyte processes. Therefore, the substantial variability of astrocyte coverage of individual hippocampal synapses implies that the efficacy of local glutamate uptake and thus the spatial fidelity of synaptic transmission is synapse dependent. By visualization of sub-diffraction-limit perisynaptic astrocytic processes and adjacent postsynaptic spines, we show that, relative to their size, small spines display a stronger coverage by astroglial transporters than bigger neighboring spines. Similarly, glutamate transients evoked by synaptic stimulation are more sensitive to pharmacological inhibition of glutamate uptake at smaller spines, whose high-affinity N-methyl-D-aspartate receptors (NMDARs) are better shielded from remotely released glutamate. At small spines, glutamate-induced and NMDAR-dependent Ca2+ entry is also more strongly increased by uptake inhibition. These findings indicate that spine size inversely correlates with the efficacy of local glutamate uptake and thereby likely determines the probability of synaptic crosstalk.

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.

Epithelial administration of low molecular weight heparin (LMWH) has proven its therapeutic efficiency in ulcerative colitis (UC) but still lacks of a sufficiently selective drug delivery system. Polymeric nanoparticles were used here not only to protect LMWH from intestinal degradation but also to provide targeted delivery to inflamed tissue in experimental colitis mice. LMWH was associated with polymethacrylate nanoparticles (NP) type A (PEMT-A) or type B (PEMT-B) of a size: 150 nm resulting in a maximum drug loading: 0.1 mg/mg. In a lipopolysaccharide-stimulated macrophages both, free LMWH and LMWH-NP have significantly reduced the cytokines secretion independently from cellular uptake. The in-vivo therapeutic efficiency was dose dependent as at low doses (100 IU/kg) only minor differences between free LMWH and LMWH-NP were found and the superiority of LMWH-NP became prominent with dose increase (500 IU/kg). Administration of LMWH-NP at 500 IU/kg has markedly improved the clinical activity as compared to LMWH while similarly pathophysiological indicators revealed increased therapeutic outcome in presence of NP compared to LMWH alone: Myeloperoxidase (Colitis control: 10 480 ± 5335, LMWH-PEMT-A NP: 1507 ± 2165, LMWH-PEMT-B NP: 382 ± 143, LMWH: 8549 ± 5021 units/g) and tumor necrosis factor: (Colitis control: 1636 ± 544, LMWH-PEMT-A NP: 511 ± 506, LMWH-PEMT-B NP: 435 ± 473, LMWH: 1110 ± 309 pg/g). Associating LMWH with NP is improving the anti-inflammatory efficiency of LMWH in-vivo by its protection against degradation in luminal environment and selective drug delivery. Such a combination holds promise for a highly specific therapy by its double selectivity towards the inflamed intestinal tissue. LMWH-PEMT NP have significantly improved the clinical activity in-vivo in comparison to free LMWH.

Within recent decades, the development of nanotechnology has made a significant contribution to the progress of various fields of study, including the domains of medical and pharmaceutical sciences. A substantially transformed arena within the context of the latter is the development and production of various injectable parenteral formulations. Indeed, recent decades have witnessed a rapid growth of the marketed and pipeline nanotechnology-based injectable products, which is a testimony to the remarkability of the aforementioned contribution. Adjunct to the ability of nanomaterials to deliver the incorporated payloads to many different targets of interest, nanotechnology has substantially assisted to the development of many further facets of the art. Such contributions include the enhancement of the drug solubility, development of long-acting locally and systemically injectable formulations, tuning the onset of the drug’s release through the endowment of sensitivity to various internal or external stimuli, as well as adjuvancy and immune activation, which is a desirable component for injectable vaccines and immunotherapeutic formulations. The current work seeks to provide a comprehensive review of all the abovementioned contributions, along with the most recent advances made within each domain. Furthermore, recent developments within the domains of passive and active targeting will be briefly debated.

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.

Synaptic signaling to NG2-expressing oligodendrocyte precursor cells (NG2 cells) could be key to rendering myelination of axons dependent on neuronal activity, but it has remained unclear whether NG2 glial cells integrate and respond to synaptic input. Here we show that NG2 cells perform linear integration of glutamatergic synaptic inputs and respond with increasing dendritic calcium elevations. Synaptic activity induces rapid Ca2+ signals mediated by low-voltage activated Ca2+ channels under strict inhibitory control of voltage-gated A-type K+ channels. Ca2+ signals can be global and originate throughout the cell. However, voltage-gated channels are also found in thin dendrites which act as compartmentalized processing units and generate local calcium transients. Taken together, the activity-dependent control of Ca2+ signals by A-type channels and the global versus local signaling domains make intracellular Ca2+ in NG2 cells a prime signaling molecule to transform neurotransmitter release into activity-dependent myelination.