Journal of Neurochemistry

The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system and mutations in Kv1.2 leads to neurological disorders such as epilepsies and ataxias. In the cerebellum regulation of Kv1.2 is coupled to learning and memory. We have previously shown that blocking Kv1.2 by infusing its specific inhibitor Tityustoxin-k (TsTX) into the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are not fully understood. We have previously shown that infusion of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the faciliatory effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression involves channel tyrosine phosphorylation and its endocytocytic removal from the cell surface. In this study we report that an excitatory chemical stimulus (50mM K+-100{micro}M glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation of mGluR1 with DHPG reduced the amount of Kv1.2 detected by cell surface biotinylation in cerebellar slices. To determine the molecular pathways involved we used an unbiased mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with PKC-{gamma}, CaMKII and Gq/G11, each of which had been shown in other studies to co-immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the interaction between Kv1.2 and PKC-{gamma} since in HEK cells and hippocampal neurons Kv1.2 endocytosis is elicited by PKC activation. Here we show that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface Kv1.2 levels elicited by DHPG, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC.

Recent work shows that certain immunological assays for the neurofilament light chain NF-L detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to NF-L, UD1 and UD2, also known as 2.1 and 47.3 respectively. These are the essential components of the Uman Diagnostics NF-Light ELISA kit, the Quanterix Simoa bead based NF-L assay and others. We show here that the antibodies bind to neighboring epitopes in a short, conserved and unusual peptide in the NF-L "rod" Coil 2 region. We also describe a surprising and useful feature of Uman and similar reagents. While other well characterized NF-L antibodies show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of presumably spontaneously degenerating or degenerated neurons and their processes. However following experimental mid-cervical injuries to rat spinal cord both Uman antibodies recognize numerous profiles in tissue sections. The Uman positive material was associated with fiber tracts expected to be damaged by the injury administered and the profiles had the swollen, beaded, discontinuous and sinusoidal morphology expected for degenerating and degenerated processes. We also found that several antibodies to the C terminal "tail" region of NF-L stain undamaged axonal profiles but fail to recognize the Uman positive material. The unmasking of the Uman epitopes and the loss of the NF-L tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunological changes we have discovered are due to neurodegeneration induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibody reagents directed against the region of NF-L including the Uman epitopes which have staining properties identical to the Uman reagents. Using these we show that the NF-L region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the NF-L biomarker, describes novel and useful properties of Uman type and NF-L tail binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

Infection and inflammation within the brain induces changes in neuronal connectivity and function. The intracellular protozoan parasite, Toxoplasma gondii, is one pathogen that infects the brain and can cause encephalitis and seizures. Persistent infection by this parasite is also associated with behavioral alterations and an increased risk for developing psychiatric illness, including schizophrenia. Current evidence from studies in humans and mouse models suggest that both seizures and schizophrenia result from a loss or dysfunction of inhibitory synapses. In line with this, we recently reported that persistent Toxoplasma gondii infection alters the distribution of glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes GABA synthesis in inhibitory synapses. These changes could reflect a redistribution of presynaptic machinery in inhibitory neurons or a loss of inhibitory nerve terminals. To directly assess the latter possibility, we employed serial block face scanning electron microscopy (SBFSEM) and quantified inhibitory perisomatic synapses in neocortex and hippocampus following parasitic infection. Not only did persistent infection lead to a significant loss of perisomatic synapses, it induced the ensheathment of neuronal somata by phagocytic cells. Immunohistochemical, genetic, and ultrastructural analyses revealed that these phagocytic cells included reactive microglia. Finally, ultrastructural analysis identified phagocytic cells enveloping perisomatic nerve terminals, suggesting they may participate in synaptic stripping. Thus, these results suggest that microglia contribute to perisomatic inhibitory synapse loss following parasitic infection and offer a novel mechanism as to how persistent Toxoplasma gondii infection may contribute to both seizures and psychiatric illness.\n\nMAIN POINTSO_LIToxoplasma-infection leads the loss of perisomatic inhibitory synapses\nC_LIO_LIPhagocytic microglia ensheath neuronal somata following Toxoplasma-infection\nC_LIO_LIMicroglia contact and envelop perisomatic nerve terminals, suggesting that Toxoplasma induces synaptic stripping\nC_LI

The mitochondrial translocator protein 18kDa (TSPO) has been linked to a variety of functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in the periphery using Leydig cells and hepatocytes, as well as work in microglia, indicate that the function of TSPO may vary between cells depending on their specialised roles. Astrocytes are critical for providing trophic and metabolic support in the brain as part of their role in maintaining brain homeostasis. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. However, relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/-) mouse primary astrocytes in vitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed metabolic flux analyses. We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO). Lactate production was significantly reduced in TSPO-/- astrocytes. Co-immunoprecipitation studies in U373 cells revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a, which presents a mechanism wherein TSPO may regulate FAO in astrocytes. Compared to TSPO+/+ cells, inflammation induced by 3h lipopolysaccharide (LPS) stimulation of TSPO-/- MPAs revealed attenuated tumour necrosis factor release, which was enhanced in TSPO-/- MPAs at 24h LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility in astrocytes, loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo. In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2s interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2s interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo. In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2s interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2s interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

Myelin is a unique, lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical components of myelin. Here we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we made measurements in isolated oligodendrocytes, the myelin-producing cells in the central nervous system. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with a focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of alternate catalytic subunits, SPTLC2 and -3, the relative levels of isoforms of the small subunits ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. As myelination progressed there were distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. The distribution of these changes among sphingolipid family members indicates that there is selective channeling of the ceramide backbone towards specific downstream metabolic pathways during myelination.

Gene expression requires two steps - transcription and translation - which can be regulated independently to allow nuanced, localized, and rapid responses to cellular stimuli. Neurons are known to respond transcriptionally and translationally to bursts of brain activity, and a transcriptional response to this activation has also been recently characterized in astrocytes. However, the extent to which astrocytes respond translationally is unknown. We tested the hypothesis that astrocytes also have a programmed translational response by characterizing the change in transcript ribosome occupancy in astrocytes using Translating Ribosome Affinity Purification(TRAP) subsequent to a robust induction of neuronal activity in vivo via acute seizure. We identified a change in transcripts on astrocyte ribosomes, highlighted by a rapid decrease in transcripts coding for ribosomal and mitochondrial components, and a rapid increase in transcripts related to cytoskeletal dynamics, motor activity, ion transport, and cell communication. This indicates a set of dynamic responses, some of which might be secondary to activation of Receptor Tyrosine Kinase(TRK) signaling. Using acute slices, we quantified the extent to which individual cues and sequela of neuronal activity can activate translation acutely in astrocytes. We identified both BDNF and ion concentration changes as contributors to translation induction, with potassium using both action-potential sensitive and insensitive components. We showed this translational response requires the presence of neurons, indicating the response is non-cell autonomous. We also show that this induction of new translation extends into peripheral astrocyte processes (PAPs). Accordingly, proteomics following fear conditioning in mice, showed that new translation influences peri-synaptic astrocyte protein composition in vivo under physiological conditions. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning. Main PointsAstrocytes have a programmed, transcript-specific translational response to neuronal activity. Both BDNF and K+, cues of neuronal activity, trigger this response. This response requires the presence of neurons. This response alters the astrocytic protein composition at the synapse.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis involves dynamic changes in glutamatergic signalling. Magnetic resonance spectroscopy can monitor these changes but lacks temporal resolution and cell-type specificity. We investigated whether urinary astrocyte-derived extracellular vesicles (ADEVs) could serve as a non-invasive proxy for brain receptor dynamics. We prospectively collected longitudinal urine and cerebrospinal fluid (CSF) samples from a 30- 35-year-old female patient during 34 days of treatment. We isolated ADEVs using a specific protocol and measured GluN1 protein levels. A 30-35-year-old healthy female provided control samples. Wavelet transform analysis of the patient's GluN1 time series revealed two distinct patterns. First, a low-frequency trend showed declining GluN1 levels over the treatment period, which mirrored the reduction in CSF GluN1 concentrations. Second, a high-frequency oscillation appeared to be coupled with methotrexate infusions, with GluN1 peaks occurring approximately 48 hours after each dose. This secondary increase may reflect drug-induced p53 activation, which promotes the exosomal release of internalised receptors. These findings suggest that urinary ADEVs provide a feasible and informative method to monitor real-time molecular fluxes in the brain.

Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) causes a systemic infection that affects the central nervous system. We used virus-like particles (VLPs) to explore how exposure to the SARS-CoV-2 proteins affects brain activity patterns in wild-type (WT) mice and in mice that express the wild-type human tau protein (htau mice). VLP exposure elicited dose-dependent changes in corticosterone and distinct chemokine levels. Longitudinal two-photon microscopy recordings of primary somatosensory and motor cortex neurons that express the jGCaMP7s calcium sensor tracked modifications of neuronal activity patterns following exposure to VLPs. There was a substantial short-term increase in stimulus-evoked activity metrics in both WT and htau VLP-injected mice, while htau mice showed also increased spontaneous activity metrics and increase activity in the vehicle-injected group. Over the following weeks, activity metrics in WT mice subsided, but remained above baseline levels. For htau mice, activity metrics either remain elevated or decreased to lower levels than baseline. Overall, our data suggest that exposure to the SARS-CoV-2 VLPs leads to strong short-term disruption of cortical activity patterns in mice with long-term residual effects. The htau mice, which have a more vulnerable genetic background, exhibited more severe pathobiology that may lead to more adverse outcomes.

Synaptic vesicles (SVs) are small organelles secreting neurotransmitters at synapses. By fusing a photoactivated fluorescent protein to VGLUT1, we generated a VGLUT1mEos2 knock-in mouse. VGLUT1mEos2 knock-in mice are viable and healthy, but exhibit a severe reduction in VGLUT1 expression levels. Using VGLUT1mEos2 expressing neurons, we established paradigms to trace individual SV mobility at the single-molecule level or via massive photoconversion. Hippocampal neurons with significantly diminished VGLUT1 expression maintain unaltered miniature glutamate release characteristics in terms of quantal size and frequency. We demonstrate that VGLUT1 expression level are not correlated in a linear fashion with the vesicular glutamate content. In conclusion, the VGLUT1mEos2 mouse line serves as a powerful tool for exploring SV mobility properties and elucidating the contributions of VGLUT1 to excitatory neurotransmission and cognitive processes.

Metals such as copper, iron, and manganese (Mn) are essential for life, but induce neurotoxicity at elevated levels. Yet, the neuronal mechanisms of metal-induced neurological disease are largely unclear. A primary limitation has been an inability to selectively alter metal levels in specific neurons, so that the role of the targeted neurons in disease biology can be isolated. Here, we show that neuron-specific depletion of metal efflux transporters provides the feasibility to overcome this limitation by focusing on Mn, which accumulates in the basal ganglia and induces motor disease, and the Mn-specific efflux transporter SLC30A10. Pan-neuronal/glial Slc30a10 knockout mice exhibited increased basal ganglia Mn levels and hypolocomotor deficits in early-life (pre-adulthood), which were exacerbated by Mn exposure. The locomotor deficits of the pan-neuronal/glial strain was associated with a reduction in evoked striatal dopamine release without dopaminergic (DAergic) neurodegeneration or changes in striatal tissue dopamine levels. Furthermore, DAergic-specific, but not GABAergic-specific, Slc30a10 knockout mice recapitulated the hypolocomotor phenotype of the pan-neuronal/glial knockouts in early-life although Mn levels were elevated in the targeted basal ganglia regions of both the neuron-specific strains. Put together, our results imply that (1) activity of SLC30A10 in DAergic neurons is necessary to protect against early-life Mn neurotoxicity; (2) increasing Mn levels in DAergic neurons is sufficient to induce early-life motor disease, suggesting that Mn targets DAergic neurons in the early-life period to induce motor deficits; and (3) neuron-specific knockout of metal efflux transporters may be a widely applicable strategy to elucidate mechanisms of metal-induced neurotoxicity.

Glucose is a predominant fuel for the brain supporting its high energy demand associated with neuronal signaling and synaptic activity. Long-term potentiation (LTP) is required for learning and memory formation by generating long lasting increase in synaptic strength and signal transmission between two neurons. While the electrophysiological bases of LTP are well established, much less is known about the metabolic demands of neurons involved in LTP. Common protocols used to examine synaptic activity rely on high glucose concentrations which are far from physiological glucose levels found in the brain. Here we used primary hippocampal neurons cultured under physiological (2.5 mM) and high (25 mM) glucose to investigate the metabolic effects of chemically induced LTP. Physiological glucose was associated with neuronal survival while high glucose promoted "PAS granule" accumulation. Changes in glucose altered extracellular lactate and pyruvate concentrations and affected key intracellular metabolic intermediates and neurotransmitter levels in neuronal cells without depleting the TCA cycle. LTP induction was comparable, but mitochondrial and neurotransmitter response to LTP was differentially affected physiological and high glucose conditions. Glycogen phosphorylase inhibition had minimal effects in physiological glucose but impaired synaptic responses and altered metabolite dynamics in high glucose. Our findings demonstrate that neuronal mitochondrial metabolism is closely linked to synaptic plasticity and highlight the importance of studying neurophysiological activity physiologically relevant glucose conditions.

The immediate-early gene Arc is a master regulator of synaptic plasticity and plays a critical role in memory consolidation. However, there has not been a comprehensive analysis of the itinerary of Arc protein, linking its function at different subcellular locations with corresponding time points after neuronal network activation. When cultured hippocampal neurons are treated with a combination of pharmacological agents to induce long term potentiation, they express high levels of Arc, allowing to study its spatiotemporal distribution. Our experiments show that neuronal activity-induced Arc expression was not restricted to neurons, but that its spatiotemporal dynamics involved a shift to astrocytes at a later timepoint. Specifically, astrocytic Arc is not due to endogenous transcription, but is dependent on the production of neuronal Arc and accumulates potentially via the recently reported intercellular transfer mechanism through Arc capsids. In conclusion, we found that Arc accumulates within astrocytes in a neuronal activity-dependent manner, which is independent of endogenous astrocytic Arc transcription, therefore highlighting the need to study the purpose of this pool of Arc, especially in learning and memory.

Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer- and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used here to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/452509v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@823562org.highwire.dtl.DTLVardef@7edb25org.highwire.dtl.DTLVardef@656feeorg.highwire.dtl.DTLVardef@197aae1_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsMACS, cytometer-FACS, and cartridge-FACS give equivalent and sufficient yield/purity for microglial analyses. Ex vivo microglial activation is prevented by supplementation with transcription/translation inhibitors during cell preparation.

Microglia, the primary immune cells of the brain, orchestrate immune responses to both external and internal stimuli in health and disease. Although several cell culture methods exist to model microglia in vitro, isolating loosely adherent microglia from primary murine mixed glial cultures remains valuable for recapitulating in vivo cell states from complex transgenic lines at relatively low cost. However, these methods are constrained by limited temporal control and scalability. To address these challenges, we established a protocol for generating microglia from frozen mixed glial cultures. While freezing introduced measurable differences in microglial morphology and lipid droplet content, microglia derived from frozen cultures responded to environmental stimuli (high glucose and LPS) in the same way as those from fresh cultures. Thus, while frozen primary microglia exhibit similar functional responses, maintaining consistency in using fresh or frozen cells within a given experiment is strongly recommended. MotivationAlthough our understanding of microglias role in brain development and disease continues to grow, a standardized in vitro model of primary mouse microglia remains absent in the field. Primary cell culture systems are essential for studying microglia, enabling the empirical assessment of gene function in transgenic models and testing novel interventions before committing to time- and cost-intensive in vivo studies. However, mixed glial culture-derived primary microglia (pMG) systems are constrained by the timing of animal availability and have limited scalability. In this study, we sought to establish a protocol for freezing and subsequently reviving primary mixed glial cultures from which responsive pMG could be isolated for downstream analysis. We validated this approach by quantifying microglia responses to distinct environmental stimuli before and after a freeze-thaw cycle. Research topicsCP: Cell biology Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/693524v2_ufig1.gif" ALT="Figure 1000"> View larger version (54K): org.highwire.dtl.DTLVardef@1b0e291org.highwire.dtl.DTLVardef@1eacfb2org.highwire.dtl.DTLVardef@e9c18aorg.highwire.dtl.DTLVardef@a151a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.

Gangliosides are sialic acid-containing glycosphingolipids highly enriched in the brain. Located mainly at the plasma membrane, gangliosides play important roles in signaling and cell-to-cell communication. Lack of gangliosides causes severe early onset neurodegenerative disorders, while more subtle deficits have been reported in Parkinsons disease and in Huntingtons disease, two misfolded protein diseases with a neuroinflammatory component. On the other hand, administration of ganglioside GM1 provides neuroprotection in both diseases and in several other models of neuronal insult. While most studies have focused on the role of endogenous gangliosides and the effects of exogenously administered GM1 in neurons, their contribution to microglia functions that are affected in neurodegenerative conditions is largely unexplored. Microglia are the immune cells of the brain and play important homeostatic functions in health and disease. In this study, we show that administration of exogenous GM1 exerts a potent anti-inflammatory effect on microglia activated with LPS, IL-1{beta} or upon phagocytosis of latex beads. These effects are partially reproduced by L-t-PDMP, a compound that stimulates the activity of the ganglioside biosynthetic pathway, while inhibition of ganglioside synthesis with GENZ-123346 increases microglial transcriptional response to LPS. We further show that administration of GM1 increases the uptake of apoptotic bodies and latex beads by microglia, as well as microglia migration and chemotaxis in response to ATP. On the contrary, decreasing microglial ganglioside levels results in a partial impairment in both microglial activities. Finally, increasing cellular ganglioside levels results in decreased expression and secretion of microglial brain derived neurotrophic factor (BDNF). Altogether, our data suggest that gangliosides are important modulators of microglia functions that are crucial to healthy brain homeostasis, and reveal that administration of ganglioside GM1 exerts an important anti-inflammatory activity that could be exploited therapeutically.

We have shown that tauopathy models display early-stage hyperexcitability due to increased presynaptic glutamate release that is mediated by an increase in vesicular glutamate transporter-1 (VGlut1). This hyperexcitability increases energy demand which in turn would increase demand on mitochondria. It is unclear, however, how early-stage presynaptic changes in glutamate release are supported by or influence the function of mitochondria. Using Large Area Scanning Electron Microscopy (LA-SEM) and fluorescence microscopy, we demonstrate that mitochondrial changes in morphology, structure, and function in CA1/CA3 hippocampal neurons decrease resting mitochondrial membrane potential in P301L mice. However, P301L mitochondria maintain a high membrane potential during levels of high activity, suggesting that they can support increased energy demand during hyperexcitability. These activity-dependent differences in membrane potential can be rescued by inhibiting ATP-dependent VGlut1 vesicle refilling. This indicates that the increased VGlut1 per vesicle observed in P301L mice contributes to the differences in mitochondria membrane potential. Notably, the mitochondrial dysfunction in P301L mice occurs before any observable alterations in presynaptic release mechanics, suggesting these changes may represent early therapeutic targets. Finally, we propose a model of increased glutamate-mediated changes in mitochondrial morphology and function in P301L neurons that represents a potentially targetable pathway to reduce or arrest neurodegeneration.

Oligodendrocyte precursor cells (OPCs) are unique glial cells that communicate bidirectionally with neurons. Neuronal inputs drive various OPC behaviors, including proliferation and differentiation, immunomodulation, blood brain barrier regulation, synapse engulfment and axonal remodeling. OPCs are implicated in numerous stress and pain conditions, where their involvement is likely driven by neuronal activity (ie. neurotransmitter and neuropeptide signaling). One neuropeptide causally involved in chronic pain and stress conditions is calcitonin gene-related peptide (CGRP). Here, we tested the hypothesis that OPCs receive direct inputs from CGRP-containing neurons in the adult brain. Using RNAscope, immunofluorescence and analysis of single-cell datasets, we find that OPCs express receptors for CGRP and we identify close spatial contacts between CGRP and OPCs, with nearly half of CGRP puncta occurring within 1 {micro}m of an OPC. Some of these contacts appear to be synaptic, with CGRP-OPC contacts colocalizing with the presynaptic protein Bassoon and the postsynaptic protein PSD-95. This work suggests the presence of both diffuse and more direct forms of CGRP signaling to OPCs, raising the importance of future experiments to identify both the mode of CGRP release onto OPCs and the functional effects of these different contact types.