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The extracellular matrix (ECM) plays a key role in synaptogenesis and the regulation of synaptic functions in the central nervous system. Recent studies revealed that in addition to dopaminergic and serotoninergic neuromodulatory systems, microglia also contribute to the regulation of ECM remodeling. In the present work, we investigated the physiological role of microglia in the remodeling of perineuronal nets (PNNs), predominantly associated with parvalbumin-immunopositive (PV+) interneurons, and the perisynaptic ECM around pyramidal neurons in the hippocampus. Adult mice were treated with PLX3397 (pexidartinib), as the inhibitor of colony-stimulating factor 1 receptor (CSF1-R), to deplete microglia. Then, confocal analysis of the ECM and synapses was performed. Although the elimination of microglia did not alter the overall number or intensity of PNNs in the CA1 region of the hippocampus, it decreased the size of PNN holes and elevated the expression of the surrounding ECM. In the neuropil area in the CA1 str. radiatum, the depletion of microglia increased the expression of perisynaptic ECM proteoglycan brevican, which was accompanied by the elevated expression of presynaptic marker vGluT1 and the increased density of dendritic spines. Thus, microglia regulate the homeostasis of pre- and postsynaptic excitatory terminals and the surrounding perisynaptic ECM as well as the fine structure of PNNs enveloping perisomatic—predominantly GABAergic—synapses.

Sporadic inclusion body myositis (sIBM) is the most prevalent acquired muscle disorder in the elderly with no defined etiology or effective therapy. Endoplasmic reticulum stress and deposition of myostatin, a secreted negative regulator of muscle growth, have been implicated in disease pathology. The myostatin signaling pathway has emerged as a major target for symptomatic treatment of muscle atrophy. Here, we systematically analyzed the maturation and secretion of myostatin precursor MstnPP and its metabolites in a human muscle cell line. We find that increased MsntPP protein levels induce ER stress. MstnPP metabolites were predominantly retained within the endoplasmic reticulum (ER), also evident in sIBM histology. MstnPP cleavage products formed insoluble high molecular weight aggregates, a process that was aggravated by experimental ER stress. Importantly, ER stress also impaired secretion of mature myostatin. Reduced secretion and aggregation of MstnPP metabolites were not simply caused by overexpression, as both events were also observed in wildtype cells under ER stress. It is tempting to speculate that reduced circulating myostatin growth factor could be one explanation for the poor clinical efficacy of drugs targeting the myostatin pathway in sIBM. Electronic supplementary material The online version of this article (10.1007/s12035-018-0997-9) contains supplementary material, which is available to authorized users.

After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of 'intermediate states' in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg-/- mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

Summary Here, we show that the lysophosphatidic acid receptor 1 (LPA1) is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways., Highlights • LPA1-GFP+ allows the prospective isolation of hippocampal precursor cells • Method for separation of proliferative from non-proliferative precursor cells • Proliferative precursor cells have a unique immune-cell-like transcriptional profile • LPA increases in vivo hippocampal neurogenesis via the LPA1-AKT and MAPK pathways, In this article, Kempermann and colleagues have identified lysophosphatidic acid receptor (LPA1) as a neural precursor cell marker. Combining expression of LPA1-GFP with EGFR and prominin-1, they have prospectively separated and subsequently transcriptionally profiled the proliferative and non-proliferative hippocampal precursor cells. In addition, they show that the corresponding ligand, LPA, is involved in the regulation of adult hippocampal neurogenesis.

The investigation of amyloid precursor protein (APP) has been mainly confined to its neuronal functions, whereas very little is known about its physiological role in astrocytes. Astrocytes exhibit a particular morphology with slender extensions protruding from somata and primary branches. Along these fine extensions, spontaneous calcium transients occur in spatially restricted microdomains. Within these microdomains mitochondria are responsible for local energy supply and Ca2+ buffering. Using two-photon in vivo Ca2+ imaging, we report a significant decrease in the density of active microdomains, frequency of spontaneous Ca2+ transients and slower Ca2+ kinetics in mice lacking APP. Mechanistically, these changes could be potentially linked to mitochondrial malfunction as our in vivo and in vitro data revealed severe, APP-dependent structural mitochondrial fragmentation in astrocytes. Functionally, such mitochondria exhibited prolonged kinetics and morphology dependent signal size of ATP-induced Ca2+ transients. Our results highlight a prominent role of APP in the modulation of Ca2+ activity in astrocytic microdomains whose precise functioning is crucial for the reinforcement and modulation of synaptic function. This study provides novel insights in APP physiological functions which are important for the understanding of the effects of drugs validated in Alzheimer's disease treatment that affect the function of APP.

Substance P (SP) is implicated in stress regulation and affective and anxiety-related behavior. Particularly high expression has been found in the main output region of the amygdala complex, the central amygdala (CE). Here we investigated the cellular mechanisms of SP in CE in vitro, taking advantage of glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knockin mice that yield a reliable labeling of GABAergic neurons, which comprise 95% of the neuronal population in the lateral section of CE (CEl). In GFP-positive neurons within CEl, SP caused a membrane depolarization and increase in input resistance, associated with an increase in action potential firing frequency. Under voltage-clamp conditions, the SP-specific membrane current reversed at −101.5 ± 2.8 mV and displayed inwardly rectifying properties indicative of a membrane K+ conductance. Moreover, SP responses were blocked by the neurokinin type 1 receptor (NK1R) antagonist L-822429 and mimicked by the NK1R agonist [Sar9,Met(O2)11]-SP. Immunofluorescence staining confirmed localization of NK1R in GFP-positive neurons in CEl, predominantly in PKCδ-negative neurons (80%) and in few PKCδ-positive neurons (17%). Differences in SP responses were not observed between the major types of CEl neurons (late firing, regular spiking, low-threshold bursting). In addition, SP increased the frequency and amplitude of GABAergic synaptic events in CEl neurons depending on upstream spike activity. These data indicate a NK1R-mediated increase in excitability and GABAergic activity in CEl neurons, which seems to mostly involve the PKCδ-negative subpopulation. This influence can be assumed to increase reciprocal interactions between CElon and CEloff pathways, thereby boosting the medial CE (CEm) output pathway and contributing to the anxiogenic-like action of SP in the amygdala.

Microglia, the central nervous system resident innate immune cells, cluster around Aβ plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aβ plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aβ plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes. In addition, patch-clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K+ currents in plaque-associated but not plaque-distant microglia. Within the subgroup of plaque-associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single-cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in- and outward K+ conductance in plaque-associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.

Innate immune cells are notoriously difficult to transfect; however, retroviruses can be used to stably integrate genes of interest into the host genome of primary or immortalized immune cells resulting in the generation of reporter cells. Here, we provide a detailed protocol covering the production of retroviruses, retroviral infection of innate immune target cells (including isolation and differentiation of murine bone marrow cells to macrophages), and several methods for enrichment of positively transduced cells.

Chemokines are important signaling molecules in the immune and nervous system. Using a fluorescence reporter mouse model, we demonstrate that the chemokine CCL17, a ligand of the chemokine receptor CCR4, is produced in the murine brain, particularly in a subset of hippocampal CA1 neurons. We found that basal expression of Ccl17 in hippocampal neurons was strongly enhanced by peripheral challenge with lipopolysaccharide (LPS). LPS-mediated induction of Ccl17 in the hippocampus was dependent on local tumor necrosis factor (TNF) signaling, whereas upregulation of Ccl22 required granulocyte-macrophage colony-stimulating factor (GM-CSF). CCL17 deficiency resulted in a diminished microglia density under homeostatic and inflammatory conditions. Further, microglia from naive Ccl17-deficient mice possessed a reduced cellular volume and a more polarized process tree as assessed by computer-assisted imaging analysis. Regarding the overall branching, cell surface area, and total tree length, the morphology of microglia from naive Ccl17-deficient mice resembled that of microglia from wild-type mice after LPS stimulation. In line, electrophysiological recordings indicated that CCL17 downmodulates basal synaptic transmission at CA3-CA1 Schaffer collaterals in acute slices from naive but not LPS-treated animals. Taken together, our data identify CCL17 as a homeostatic and inducible neuromodulatory chemokine affecting the presence and morphology of microglia and synaptic transmission in the hippocampus.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

One of the hallmarks of Alzheimer's disease is the formation of neurofibrillary tangles, intracellular aggregates of hyperphosphorylated, mislocalized tau protein, which are associated with neuronal loss. Changes in tau are known to impair cellular transport (including that of mitochondria) and are associated with cell death in cell culture and mouse models of tauopathy. Thus clearing pathological forms of tau from cells is a key therapeutic strategy. One critical modulator in the degradation and clearance of misfolded proteins is the co-chaperone CHIP (Carboxy terminus Hsp70 interacting Protein), which is known to play a role in refolding and clearance of hyperphosphorylated tau. Here, we tested the hypothesis that CHIP could ameliorate pathological changes associated with tau. We find that co-expressing CHIP with full-length tau, tau truncated at D421 mimicking caspase cleavage, or the short tauRDΔK280 tau construct containing only the tau repeat domain with a tauopathy mutation, decreases tau protein levels in human H4 neuroglioma cells in a manner dependent on the Hsp70-binding TPR domain of CHIP. The observed reduction in tau levels by CHIP is associated with a decrease of tau phosphorylation and reduced levels of cleaved Caspase 3 indicating that CHIP plays an important role in preventing tau-induced pathological changes. Furthermore, tau-associated mitochondrial transport deficits are rescued by CHIP co-expression in H4 cells. Together, these data suggest that the co-chaperone CHIP can rescue the pathological effects of tau, and indicate that other diseases of protein misfolding and accumulation may also benefit from CHIP upregulation.

Optimal reproductive fitness is essential for the biological success and survival of species. The vomeronasal organ is strongly implicated in the display of sexual and reproductive behaviors in female mice, yet the roles that apical and basal vomeronasal neuron populations play in controlling these gender-specific behaviors remain largely unclear. Results To dissect the neural pathways underlying these functions, we genetically inactivated the basal vomeronasal organ layer using conditional, cell-specific ablation of the G protein Gαo. Female mice mutant for Gαo show severe alterations in sexual and reproductive behaviors, timing of puberty onset, and estrous cycle. These mutant mice are insensitive to reproductive facilitation stimulated by male pheromones that accelerate puberty and induce ovulation. Gαo-mutant females exhibit a striking reduction in sexual receptivity or lordosis behavior to males, but gender discrimination seems to be intact. These mice also show a loss in male scent preference, which requires a learned association for volatile olfactory signals with other nonvolatile ownership signals that are contained in the high molecular weight fraction of male urine. Thus, Gαo impacts on both instinctive and learned social responses to pheromones. Conclusions These results highlight that sensory neurons of the Gαo-expressing vomeronasal subsystem, together with the receptors they express and the molecular cues they detect, control a wide range of fundamental mating and reproductive behaviors in female mice. Fil: Oboti, Livio. University of Saarland School of Medicine. Hamburgo; Alemania. Center for Neuroscience Research. Washington; Estados Unidos Fil: Pérez Gómez, Anabel. University of Saarland School of Medicine. Hamburgo; Alemania Fil: Keller, Matthieu. Universite de Tours; Francia Fil: Jacobi, Eric. University of Saarland School of Medicine. Hamburgo; Alemania. Deutsches Zentrum für Neurodegenerative Erkrankunge. Heidelberg; Armenia Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institute of Environmental Health Sciences; Estados Unidos Fil: Leinders Zufall, Trese. University of Saarland School of Medicine. Hamburgo; Alemania Fil: Zufall, Frank. University of Saarland School of Medicine. Hamburgo; Alemania Fil: Chamero, Pablo. University of Saarland School of Medicine. Hamburgo; Alemania

GABA(A) receptors (GABA(A)Rs) are hetero-pentameric chloride channels and the primary sites for fast synaptic inhibition. We have expressed recombinant γ2 and δ subunits of GABA(A)Rs in cultured hippocampal neurons to analyze the membrane targeting of synaptic and extra-synaptic GABA(A)Rs, a phenomenon not well understood. Our data demonstrate that the synaptic targeting of γ2-containing GABA(A)Rs (γ2-GABA(A)Rs) does not depend on the cytoplasmic loop of γ2 subunit, in parallel with previous findings, showing that the synaptic localization of γ2-GABA(A)Rs requires the TM4 domain of γ2 rather than the large cytoplasmic loop. On the other hand, we showed here that the extrasynaptic targeting of the δ-containing GABA(A)Rs (δ-GABA(A)Rs) depends on the cytoplasmic loop of δ subunit via an active or a passive mechanism. We also show that the amino acid sequences of δ loop is highly conserved across the whole span of vertebrate evolution suggesting an active role of δ loop in extra-synaptic targeting of corresponding receptor subtypes.

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson's disease (PD). The present study examined delivery of bone marrow-derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD [(Thy1)-h[A30P] aS] and an APP/PS1 model of Alzheimer's disease (AD) via intranasal application (INA). INA of microglia in naive BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1 × 104) after INA of 1 × 106cells, while the total amount of cells detected in peripheral organs did not exceed 3.4 × 103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] aS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13-month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular amyloid β (macrophages in APP/PS1 model) or α-synuclein [MSCs in (Thy1)-h[A30P] aS model] immunoreactivity. Here, we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals.

BACKGROUND: Deregulation of the protein kinase MARK has been linked to Alzheimer disease. - RESULTS: Mark-specific inhibitors and a biosensor are identified. - CONCLUSION: The inhibitors and the biosensor are tools to provide new insights into the role of MARK during polarity establishment and maintenance of neurons. - SIGNIFICANCE: The inhibitors might possess therapeutic potential by interfering with abnormal Tau phosphorylation in Alzheimer disease. Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.