hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons.

Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X-/- knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X-/- astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD.

Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

Origin of hydroxyl pair formation on reduced anatase TiO2(101).

The interaction of water with metal oxide surfaces is of key importance to several research fields and applications. Because of its ability to photo-catalyze water splitting, reducible anatase TiO2 (a-TiO2) is of particular interest. Here, we combine experiments and theory to study the dissociation of water on bulk-reduced a-TiO2(101). Following large water exposures at room temperature, point-like protrusions appear on the a-TiO2(101) surface, as shown by scanning tunneling microscopy (STM). These protrusions originate from hydroxyl pairs, consisting of terminal and bridging OH groups, OHt/OHb, as revealed by infrared reflection absorption spectroscopy (IRRAS) and valence band experiments. Utilizing density functional theory (DFT) calculations, we offer a comprehensive model of the water/a-TiO2(101) interaction. This model also explains why the hydroxyl pairs are thermally stable up to ∼480 K.

The VGF-derived Peptide TLQP21 Impairs Purinergic Control of Chemotaxis and Phagocytosis in Mouse Microglia.

Microglial cells are considered as sensors of brain pathology by detecting any sign of brain lesions, infections, or dysfunction and can influence the onset and progression of neurological diseases. They are capable of sensing their neuronal environment via many different signaling molecules, such as neurotransmitters, neurohormones and neuropeptides. The neuropeptide VGF has been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide and has been shown to signal via C3aR1 and C1qBP receptors. The effect of TLQP21 on microglial functions in health or disease is not known. Studying microglial cells in acute brain slices, we found that TLQP21 impaired metabotropic purinergic signaling. Specifically, it attenuated the ATP-induced activation of a K+ conductance, the UDP-stimulated phagocytic activity, and the ATP-dependent laser lesion-induced process outgrowth. These impairments were reversed by blocking C1qBP, but not C3aR1 receptors. While microglia in brain slices from male mice lack C3aR1 receptors, both receptors are expressed in primary cultured microglia. In addition to the negative impact on purinergic signaling, we found stimulating effects of TLQP21 in cultured microglia, which were mediated by C3aR1 receptors: it directly evoked membrane currents, stimulated basal phagocytic activity, evoked intracellular Ca2+ transient elevations, and served as a chemotactic signal. We conclude that TLQP21 has differential effects on microglia depending on C3aR1 activation or C1qBP-dependent attenuation of purinergic signaling. Thus, TLQP21 can modulate the functional phenotype of microglia, which may have an impact on their function in health and disease.SIGNIFICANCE STATEMENT The neuropeptide VGF and its peptides have been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide that activates C1qBP receptors, which are expressed by microglia. We show here, for the first time, that TLQP21 impairs P2Y-mediated purinergic signaling and related functions. These include modulation of phagocytic activity and responses to injury. As purinergic signaling is central for microglial actions in the brain, this TLQP21-mediated mechanism might regulate microglial activity in health and disease. We furthermore show that, in addition to C1qBP, functional C3aR1 responses contribute to TLQP21 action on microglia. However, C3aR1 responses were only present in primary cultures but not in situ, suggesting that the expression of these receptors might vary between different microglial activation states.

Adsorption and reaction of methanol on Fe3O4(001).

The interaction of methanol with iron oxide surfaces is of interest due to its potential in hydrogen storage and from a fundamental perspective as a chemical probe of reactivity. We present here a study examining the adsorption and reaction of methanol on magnetite Fe3O4(001) at cryogenic temperatures using a combination of temperature programmed desorption, x-ray photoelectron spectroscopy, and scanning tunneling microscopy. The methanol desorption profile from Fe3O4(001) is complex, exhibiting peaks at 140 K, 173 K, 230 K, and 268 K, corresponding to the desorption of intact methanol, as well as peaks at 341 K and 495 K due to the reaction of methoxy intermediates. The saturation of a monolayer of methanol corresponds to ∼5 molecules/unit cell (u.c.), which is slightly higher than the number of surface octahedral iron atoms of 4/u.c. We probe the kinetics and thermodynamics of the desorption of molecular methanol using inversion analysis. The deconvolution of the complex desorption profile into individual peaks allows for calculations of both the desorption energy and the prefactor of each feature. The initial 0.7 methanol/u.c. reacts to form methoxy and hydroxy intermediates at 180 K, which remain on the surface above room temperature after intact methanol has desorbed. The methoxy species react via one of two channels, a recombination reaction with surface hydroxyls to form additional methanol at ∼350 K and a disproportionation reaction to form methanol and formaldehyde at ∼500 K. Only 20% of the methoxy species undergo the disproportionation reaction, with most of them reacting via the 350 K pathway.

Water Dissociation and Hydroxyl Ordering on Anatase TiO_{2}(001)-(1×4).

We studied the interaction of water with the anatase TiO_{2}(001) surface by means of scanning tunneling microscopy, x-ray photoelectron spectroscopy, and density functional theory calculations. Water adsorbs dissociatively on the ridges of a (1×4) reconstructed surface, resulting in a (3×4) periodic structure of hydroxyl pairs. We observed this process at 120 K, and the created hydroxyls desorb from the surface by recombination to water, which occurs below 300 K. Our calculations reveal the water dissociation mechanism and uncover a very pronounced dependence on the coverage. This strong coverage dependence is explained through water-induced reconstruction on anatase TiO_{2}(001)-(1×4). The high intrinsic reactivity of the anatase TiO_{2}(001) surface towards water observed here is fundamentally different from that seen on other surfaces of titania and may explain its high catalytic activity in heterogeneous catalysis and photocatalysis.

NH3 adsorption on anatase-TiO2(101).

The adsorption of ammonia on anatase TiO2 is of fundamental importance for several catalytic applications of TiO2 and for probing acid-base interactions. Utilizing high-resolution scanning tunneling microscopy (STM), synchrotron X-ray photoelectron spectroscopy, temperature-programmed desorption (TPD), and density functional theory (DFT), we identify the adsorption mode and quantify the adsorption strength on the anatase TiO2(101) surface. It was found that ammonia adsorbs non-dissociatively as NH3 on regular five-fold coordinated titanium surface sites (5f-Ti) with an estimated exothermic adsorption energy of 1.2 eV for an isolated ammonia molecule. For higher adsorbate coverages, the adsorption energy progressively shifts to smaller values, due to repulsive intermolecular interactions. The repulsive adsorbate-adsorbate interactions are quantified using DFT and autocorrelation analysis of STM images, which both showed a repulsive energy of ∼50 meV for nearest neighbor sites and a lowering in binding energy for an ammonia molecule in a full monolayer of 0.28 eV, which is in agreement with TPD spectra.

Experimental Cortical Spreading Depression Induces NMDA Receptor Dependent Potassium Currents in Microglia.

UNLABELLED: Cortical spreading depression (CSD) is a propagating event of neuronal depolarization, which is considered as the cellular correlate of the migraine aura. It is characterized by a change in the intrinsic optical signal and by a negative DC potential shift. Microglia are the resident macrophages of the CNS and act as sensors for pathological changes. In the present study, we analyzed whether microglial cells might sense CSD by recording membrane currents from microglia in acutely isolated cortical mouse brain slices during an experimentally induced CSD. Coincident with the change in the intrinsic optical signal and the negative DC potential shift we recorded an increase in potassium conductance predominantly mediated by K(+) inward rectifier (Kir)2.1, which was blocked by the NMDA receptor antagonist D-AP5. Application of NMDA and an increase in extracellular K(+) mimics the CSD-induced Kir activation. Application of D-AP5, but not the purinergic receptor antagonist RB2, blocks the NMDA-induced Kir activation. The K(+) channel blocker Ba(2+) blocks both the CSD- and the NMDA-triggered increase in Kir channel activity. In addition, we could confirm previous findings that microglia in the adult brain do not express functional NMDA receptors by recording from microglia cultured from adult brain. From these observations we conclude that CSD activates neuronal NMDA receptors, which lead to an increase in extracellular [K(+)] resulting in the activation of Kir channel activity in microglia. SIGNIFICANCE STATEMENT: Cortical spreading depression (CSD) is a wave of neuronal depolarization spreading through the cortex and is associated with the aura of migraine. Here we show that microglial cells, which are viewed as pathologic sensors of the brain, can sense this wave. The increase in the extracellular potassium concentration associated with that wave leads to the activation of an inward rectifying potassium conductance in microglia. The involvement of neuronal NMDA receptors is crucial because NMDA mimics that response and microglia do not express functional NMDA receptors. Although it is now evident that CSD leads to a signal in microglia, the consequences of this microglial activation during CSD needs to be explored.

Facile embedding of single vanadium atoms at the anatase TiO2(101) surface.

To understand the structure-reactivity relationships for mixed-metal oxide catalysts, well-defined systems are required. Mixtures of vanadia and titania (TiO2) are of particular interest for application in heterogeneous catalysis, with TiO2 often acting as the support. By utilizing high-resolution scanning tunneling microscopy, we studied the interaction of vanadium (V) with the anatase TiO2(101) surface in the sub-monolayer regime. At 80 K, metallic V nucleates into homogeneously distributed clusters onto the terraces with no preference for nucleation at the step edges. However, embedding of single V atoms into TiO2 occurs following annealing at room temperature. In conjunction with X-ray photoelectron spectroscopy data and density functional theory calculations, we propose that monomeric V atoms occupy positions of regular surface Ti sites, i.e., Ti atoms are substituted by V atoms.

Satellite microglia show spontaneous electrical activity that is uncorrelated with activity of the attached neuron.

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact, and biocytin injection into satellite microglia showed no dye-coupling with the apposed neurons or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations that were not correlated with neuronal activity. These events could be divided into fast-rising and slow-rising depolarizations, which showed different characteristics in satellite and non-satellite microglia. Fast-rising and slow-rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast-rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.

Formation and sintering of Pt nanoparticles on vicinal rutile TiO2 surfaces.

By means of scanning tunnelling microscopy (STM) the nucleation, growth and sintering of platinum nanoparticles (Pt NP's) was studied on vicinal and flat rutile titanium dioxide (TiO2) surfaces. Utilising physical vapour deposition, the nucleation of Pt NP's on TiO2 surfaces at room temperature (RT) was found to be random and invariant towards different surface morphologies and reduction states. Thus, the nucleation of Pt on TiO2 at RT is rather insensitive to the surface structure and surface defects. Vacuum-annealing at 600 K, 700 K and 800 K, respectively, led to lower densities of Pt NP's as a result of sintering. Sintering occurred at different rates at the TiO2 surfaces studied, indicating that the surface morphology and the amount of Ti(3+) excess charge do have an influence on the particle stability. Observed changes in the NP distribution as a result of sintering can be explained inferring facile diffusion of Pt NP's along the [001] direction.

A microglia-containing cerebral organoid model to study early life immune challenges.

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.

Simultaneous imaging of redox states in dystrophic neurites and microglia at Aß plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress.

The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer's Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aß plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aß plaques but instead create protective barriers around Aß plaques possibly reducing the spread of Aß. Intracranial injection of Aß was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia. In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aß plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs.

The importance of bulk Ti3+ defects in the oxygen chemistry on titania surfaces.

The role of bulk defects in the oxygen chemistry on reduced rutile TiO(2)(110)-(1 × 1) has been studied by means of temperature-programmed desorption spectroscopy and scanning tunneling microscopy measurements. Following O(2) adsorption at 130 K, the amount of O(2) desorbing at ∼410 K initially increased with increasing density of surface oxygen vacancies but decreased after further reduction of the TiO(2)(110) crystal. We explain these results by withdrawal of excess charge (Ti(3+)) from the TiO(2)(110) lattice to oxygen species on the surface and by a reaction of Ti interstitials with O adatoms upon heating. Important consequences for the understanding of the O(2)-TiO(2) interaction are discussed.

5G connected and automated driving: use cases, technologies and trials in cross-border environments.

Cooperative, connected and automated mobility (CCAM) across Europe requires harmonized solutions to support cross-border seamless operation. The possibility of providing CCAM services across European countries has an enormous innovative business potential. However, the seamless provision of connectivity and the uninterrupted delivery of real-time services pose technical challenges which 5G technologies aim to solve. The situation is particularly challenging given the multi-country, multi-operator, multi-telco-vendor, multi-car-manufacturer and cross-network-generation scenario of any cross-border scenario. Motivated by this, the 5GCroCo project, with a total budget of 17 million Euro and partially funded by the European Commission, aims at validating 5G technologies in the Metz-Merzig-Luxembourg cross-border 5G corridor considering the borders between France, Germany and Luxembourg. The activities of 5GCroCo are organized around three use cases: (1) Tele-operated Driving, (2) high-definition map generation and distribution for automated vehicles and (3) Anticipated Cooperative Collision Avoidance (ACCA). The results of the project help contribute to a true European transnational CCAM. This paper describes the overall objectives of the project, motivated by the discussed challenges of cross-border operation, the use cases along with their requirements, the technical 5G features that will be validated and provides a description of the planned trials within 5GCroCo together with some initial results.

Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

Atomic-Scale View of the Oxidation and Reduction of Supported Ultrathin FeO Islands.

By means of scanning tunneling microscopy (STM) measurements, we studied in situ the oxidation and reduction of FeO bilayer islands on Au(111) by oxygen (O2) and hydrogen (H2), respectively. The FeO islands respond very dynamically toward O2, with the coordinatively unsaturated ferrous (CUF) sites at the island edges being essential for O2 dissociation and O atom incorporation. An STM movie obtained during oxidation reveals how further O2 molecules can dissociate after the consumption of all initially existing CUF sites through the formation of new CUF sites. In contrast, we found that H2 molecules only dissociate when vibrationally excited through the ion gauge and only at the basal plane of FeO islands, implying that the CUF sites are not relevant for H2 dissociation. Our STM results reveal how excess O atoms are incorporated and released in O2 and H2 and thus shed light onto the stability of inverse catalysts during a catalyzed reaction.

The adenosine generating enzymes CD39/CD73 control microglial processes ramification in the mouse brain.

Microglial cells invade the brain as amoeboid precursors and acquire a highly ramified morphology in the postnatal brain. Microglia express all essential purinergic elements such as receptors, nucleoside transporters and ecto-enzymes, including CD39 (NTPDase1) and CD73 (5'-nucleotidase), which sequentially degrade extracellular ATP to adenosine. Here, we show that constitutive deletion of CD39 and CD73 or both caused an inhibition of the microglia ramified phenotype in the brain with a reduction in the length of processes, branching frequency and number of intersections with Sholl spheres. In vitro, unlike wild-type microglia, cd39-/- and cd73-/- microglial cells were less complex and did not respond to ATP with the transformation into a more ramified phenotype. In acute brain slices, wild-type microglia retracted approximately 50% of their processes within 15 min after slicing of the brain, and this phenomenon was augmented in cd39-/- mice; moreover, the elongation of microglial processes towards the source of ATP or towards a laser lesion was observed only in wild-type but not in cd39-/- microglia. An elevation of extracellular adenosine 1) by the inhibition of adenosine transport with dipyridamole, 2) by application of exogenous adenosine or 3) by degradation of endogenous ATP/ADP with apyrase enhanced spontaneous and ATP-induced ramification of cd39-/- microglia in acute brain slices and facilitated the transformation of cd39-/- and cd73-/- microglia into a ramified process-bearing phenotype in vitro. These data indicate that under normal physiological conditions, CD39 and CD73 nucleotidases together with equilibrative nucleoside transporter 1 (ENT1) control the fate of extracellular adenosine and thereby the ramification of microglial processes.