NECAB family of neuronal calcium-binding proteins in health and disease.

The N-terminal EF-hand calcium-binding proteins 1-3 (NECAB1-3) constitute a family of predominantly neuronal proteins characterized by the presence of at least one EF-hand calcium-binding domain and a functionally less well characterized C-terminal antibiotic biosynthesis monooxygenase domain. All three family members were initially discovered due to their interactions with other proteins. NECAB1 associates with synaptotagmin-1, a critical neuronal protein involved in membrane trafficking and synaptic vesicle exocytosis. NECAB2 interacts with predominantly striatal G-protein-coupled receptors, while NECAB3 partners with amyloid-ß A4 precursor protein-binding family A members 2 and 3, key regulators of amyloid-ß production. This demonstrates the capacity of the family for interactions with various classes of proteins. NECAB proteins exhibit distinct subcellular localizations: NECAB1 is found in the nucleus and cytosol, NECAB2 resides in endosomes and the plasma membrane, and NECAB3 is present in the endoplasmic reticulum and Golgi apparatus. The antibiotic biosynthesis monooxygenase domain, an evolutionarily ancient component, is akin to atypical heme oxygenases in prokaryotes but is not well-characterized in vertebrates. Prokaryotic antibiotic biosynthesis monooxygenase domains typically form dimers, suggesting that calcium-mediated conformational changes in NECAB proteins may induce antibiotic biosynthesis monooxygenase domain dimerization, potentially activating some enzymatic properties. However, the substrate for this enzymatic activity remains uncertain. Alternatively, calcium-mediated conformational changes might influence protein interactions or the subcellular localization of NECAB proteins by controlling the availability of protein-protein interaction domains situated between the EF hands and the antibiotic biosynthesis monooxygenase domain. This review summarizes what is known about genomic organization, tissue expression, intracellular localization, interaction partners, and the physiological and pathophysiological role of the NECAB family.

Selective neuronal expression of progranulin is sufficient to provide neuroprotective and anti-inflammatory effects after traumatic brain injury.

Progranulin (PGRN), which is produced in neurons and microglia, is a neurotrophic and anti-inflammatory glycoprotein. Human loss-of-function mutations cause frontotemporal dementia, and PGRN knockout (KO) mice are a model for dementia. In addition, PGRN KO mice exhibit severe phenotypes in models of traumatic or ischemic central nervous system (CNS) disorders, including traumatic brain injury (TBI). It is unknown whether restoration of progranulin expression in neurons (and not in microglia) might be sufficient to prevent excessive TBI-evoked brain damage. To address this question, we generated mice with Nestin-Cre-driven murine PGRN expression in a PGRN KO line (PGRN-KONestinGrn) to rescue PGRN in neurons. PGRN expression analysis in primary CNS cell cultures from naïve mice and in (non-) injured brain tissue from PGRN-KONestinGrn revealed expression of PGRN in neurons but not in microglia. After experimental TBI, examination of the structural brain damage at 5 days post-injury (dpi) showed that the TBI-induced loss of brain tissue and hippocampal neurons was exacerbated in PGRN-KOGrnflfl mice (PGRN knockout with the mGrn fl-STOP-fl allele, Cre-negative), as expected, whereas the tissue damage in PGRN-KONestinGrn mice was similar to that in PGRN-WT mice. Analysis of CD68+ immunofluorescent microglia and Cd68 mRNA expression showed that excessive microglial activation was rescued in PGRN-KONestinGrn mice, and the correlation of brain injury with Cd68 expression suggested that Cd68 was a surrogate marker for excessive brain injury caused by PGRN deficiency. The results show that restoring neuronal PGRN expression was sufficient to rescue the exacerbated neuropathology of TBI caused by PGRN deficiency, even in the absence of microglial PGRN. Hence, endogenous microglial PGRN expression was not essential for the neuroprotective or anti-inflammatory effects of PGRN after TBI in this study.

The IgLON family of cell adhesion molecules expressed in developing neural circuits ensure the proper functioning of the sensory system in mice.

Deletions and malfunctions of the IgLON family of cell adhesion molecules are associated with anatomical, behavioral, and metabolic manifestations of neuropsychiatric disorders. We have previously shown that IgLON genes are expressed in sensory nuclei/pathways and that IgLON proteins modulate sensory processing. Here, we examined the expression of IgLON alternative promoter-specific isoforms during embryonic development and studied the sensory consequences of the anatomical changes when one of the IgLON genes, Negr1, is knocked out. At the embryonal age of E12.5 and E13.5, various IgLONs were distributed differentially and dynamically in the developing sensory areas within the central and peripheral nervous system, as well as in limbs and mammary glands. Sensory tests showed that Negr1 deficiency causes differences in vestibular function and temperature sensitivity in the knockout mice. Sex-specific differences were noted across olfaction, vestibular functioning, temperature regulation, and mechanical sensitivity. Our findings highlight the involvement of IgLON molecules during sensory circuit formation and suggest Negr1's critical role in somatosensory processing.

Nebulized Lipopolysaccharide Causes Delayed Cortical Neuroinflammation in a Murine Model of Acute Lung Injury.

Lung injury caused by respiratory infection is a major cause of hospitalization and mortality and a leading origin of sepsis. Sepsis-associated encephalopathy and delirium are frequent complications in patients with severe lung injury, yet the pathogenetic mechanisms remain unclear. Here, 70 female C57BL/6 mice were subjected to a single full-body-exposure with nebulized lipopolysaccharide (LPS). Neuromotor impairment was assessed repeatedly and brain, blood, and lung samples were analyzed at survival points of 24 h, 48 h, 72 h, and 96 h after exposure. qRT-PCR revealed increased mRNA-expression of TNFα and IL-1ß 24 h and 48 h after LPS-exposure in the lung, concomitantly with increased amounts of proteins in bronchoalveolar lavage and interstitial lung edema. In the cerebral cortex, at 72 h and/or 96 h after LPS exposure, the inflammation- and activity-associated markers TLR4, GFAP, Gadd45b, c-Fos, and Arc were increased. Therefore, single exposure to nebulized LPS not only triggers an early inflammatory reaction in the lung but also induces a delayed neuroinflammatory response. The identified mechanisms provide new insights into the pathogenesis of sepsis-associated encephalopathy and might serve as targets for future therapeutic approaches.

Sortilin is dispensable for secondary injury processes following traumatic brain injury in mice.

Traumatic brain injury (TBI) is characterized by complex secondary injury processes involving the p75 neurotrophin receptor (p75NTR), which has been proposed as a possible therapeutic target. However, the pathogenic role of the p75NTR co-receptor sortilin in TBI has not been investigated. In this study, we examined whether sortilin contributes to acute and early processes of secondary injury using a murine controlled cortical impact (CCI) model of TBI. Initial expression analysis showed a down-regulation of sortilin mRNA levels 1 and 5 day post injury (dpi) and a reduced expression of sortilin protein 1 dpi. Next, a total of 40 SortilinΔExon14 loss-of-function mouse mutants (Sort1-/-) and wild-type (Sort1+/+) littermate mice were subjected to CCI and examined at 1 and 5 dpi. Neither sensorimotor deficits or brain lesion size nor CCI-induced cell death or calcium-dependent excitotoxicity as evaluated by TUNEL staining or Western blot analysis of alpha II spectrin breakdown products were different between Sort1-/- and Sort1+/+ mice. In addition, CCI induced the up-regulation of pro-inflammatory marker mRNA expression (Il6, Tnfa, Aif1, and Gfap) irrespectively of the genotype. Similarly, the mRNA expressions of neurotrophins (Bdnf, Ngf, Nt3), VPS10P domain receptors others than sortilin (Ngfr, Sorl1, Sorcs2), and the sortilin interactor progranulin were not affected by genotype. Our results suggest that sortilin is a modulatory rather than a critical factor in the acute and early brain tissue response after TBI.

Brain-Bone Crosstalk in a Murine Polytrauma Model Promotes Bone Remodeling but Impairs Neuromotor Recovery and Anxiety-Related Behavior.

Traumatic brain injury (TBI) and long bone fractures are a common injury pattern in polytrauma patients and modulate each other's healing process. As only a limited number of studies have investigated both traumatic sites, we tested the hypothesis that brain-bone polytrauma mutually impacts neuro- and osteopathological outcomes. Adult female C57BL/6N mice were subjected to controlled cortical impact (CCI), and/or osteosynthetic stabilized femoral fracture (FF), or sham surgery. Neuromotor and behavioral impairments were assessed by neurological severity score, open field test, rotarod test, and elevated plus maze test. Brain and bone tissues were processed 42 days after trauma. CCI+FF polytrauma mice had increased bone formation as compared to FF mice and increased mRNA expression of bone sialoprotein (BSP). Bone fractures did not aggravate neuropathology or neuroinflammation assessed by cerebral lesion size, hippocampal integrity, astrocyte and microglia activation, and gene expression. Behavioral assessments demonstrated an overall impaired recovery of neuromotor function and persistent abnormalities in anxiety-related behavior in polytrauma mice. This study shows enhanced bone healing, impaired neuromotor recovery and anxiety-like behavior in a brain-bone polytrauma model. However, bone fractures did not aggravate TBI-evoked neuropathology, suggesting the existence of outcome-relevant mechanisms independent of the extent of brain structural damage and neuroinflammation.

Valproic Acid Treatment after Traumatic Brain Injury in Mice Alleviates Neuronal Death and Inflammation in Association with Increased Plasma Lysophosphatidylcholines.

The histone deacetylase inhibitor (HDACi) valproic acid (VPA) has neuroprotective and anti-inflammatory effects in experimental traumatic brain injury (TBI), which have been partially attributed to the epigenetic disinhibition of the transcription repressor RE1-Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). Additionally, VPA changes post-traumatic brain injury (TBI) brain metabolism to create a neuroprotective environment. To address the interconnection of neuroprotection, metabolism, inflammation and REST/NRSF after TBI, we subjected C57BL/6N mice to experimental TBI and intraperitoneal VPA administration or vehicle solution at 15 min, 1, 2, and 3 days post-injury (dpi). At 7 dpi, TBI-induced an up-regulation of REST/NRSF gene expression and HDACi function of VPA on histone H3 acetylation were confirmed. Neurological deficits, brain lesion size, blood-brain barrier permeability, or astrogliosis were not affected, and REST/NRSF target genes were only marginally influenced by VPA. However, VPA attenuated structural damage in the hippocampus, microgliosis and expression of the pro-inflammatory marker genes. Analyses of plasma lipidomic and polar metabolomic patterns revealed that VPA treatment increased lysophosphatidylcholines (LPCs), which were inversely associated with interleukin 1 beta (Il1b) and tumor necrosis factor (Tnf) gene expression in the brain. The results show that VPA has mild neuroprotective and anti-inflammatory effects likely originating from favorable systemic metabolic changes resulting in increased plasma LPCs that are known to be actively taken up by the brain and function as carriers for neuroprotective polyunsaturated fatty acids.

Current state of neuroprotective therapy using antibiotics in human traumatic brain injury and animal models.

TBI is a leading cause of death and disability in young people and older adults worldwide. There is no gold standard treatment for TBI besides surgical interventions and symptomatic relief. Post-injury infections, such as lower respiratory tract and surgical site infections or meningitis are frequent complications following TBI. Whether the use of preventive and/or symptomatic antibiotic therapy improves patient mortality and outcome is an ongoing matter of debate. In contrast, results from animal models of TBI suggest translational perspectives and support the hypothesis that antibiotics, independent of their anti-microbial activity, alleviate secondary injury and improve neurological outcomes. These beneficial effects were largely attributed to the inhibition of neuroinflammation and neuronal cell death. In this review, we briefly outline current treatment options, including antibiotic therapy, for patients with TBI. We then summarize the therapeutic effects of the most commonly tested antibiotics in TBI animal models, highlight studies identifying molecular targets of antibiotics, and discuss similarities and differences in their mechanistic modes of action.

Repurposing of pexidartinib for microglia depletion and renewal.

Pexidartinib (PLX3397) is a small molecule receptor tyrosine kinase inhibitor of colony stimulating factor 1 receptor (CSF1R) with moderate selectivity over other members of the platelet derived growth factor receptor family. It is approved for treatment of tenosynovial giant cell tumors (TGCT). CSF1R is highly expressed by microglia, which are macrophages of the central nervous system (CNS) that defend the CNS against injury and pathogens and contribute to synapse development and plasticity. Challenged by pathogens, apoptotic cells, debris, or inflammatory molecules they adopt a responsive state to propagate the inflammation and eventually return to a homeostatic state. The phenotypic switch may fail, and disease-associated microglia contribute to the pathophysiology in neurodegenerative or neuropsychiatric diseases or long-lasting detrimental brain inflammation after brain, spinal cord or nerve injury or ischemia/hemorrhage. Microglia also contribute to the growth permissive tumor microenvironment of glioblastoma (GBM). In rodents, continuous treatment for 1-2 weeks via pexidartinib food pellets leads to a depletion of microglia and subsequent repopulation from the remaining fraction, which is aided by peripheral monocytes that search empty niches for engraftment. The putative therapeutic benefit of such microglia depletion or forced renewal has been assessed in almost any rodent model of CNS disease or injury or GBM with heterogeneous outcomes, but a tendency of partial beneficial effects. So far, microglia monitoring e.g. via positron emission imaging is not standard of care for patients receiving Pexidartinib (e.g. for TGCT), so that the depletion and repopulation efficiency in humans is still largely unknown. Considering the virtuous functions of microglia, continuous depletion is likely no therapeutic option but short-lasting transient partial depletion to stimulate microglia renewal or replace microglia in genetic disease in combination with e.g. stem cell transplantation or as part of a multimodal concept in treatment of glioblastoma appears feasible. The present review provides an overview of the preclinical evidence pro and contra microglia depletion as a therapeutic approach.

NECAB2 is an endosomal protein important for striatal function.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Pre-traumatic antibiotic-induced microbial depletion reduces neuroinflammation in acute murine traumatic brain injury.

The connection between dysbiosis of the gut microbiome and diseases and injuries of the brain has attracted considerable interest in recent years. Interestingly, antibiotic-induced microbial dysbiosis has been implicated in the pathogenesis of traumatic brain injury (TBI), while early administration of antibiotics associates with improved survival in TBI patients. In animal models of TBI, short- or long-term administration of antibiotics, both peri- or post-operatively, were shown to induce gut microbiome dysbiosis but also anti-inflammatory and neuroprotective effects. However, the acute consequences of microbial dysbiosis on TBI pathogenesis after discontinuation of antibiotic treatment are elusive. In this study, we tested whether pre-traumatic antibiotic-induced microbial depletion by vancomycin, amoxicillin, and clavulanic acid affects pathogenesis during the acute phase of TBI in adult male C57BL/6 mice. Pre-traumatic microbiome depletion did not affect neurological deficits over 72 h post injury (hpi) and brain histopathology, including numbers of activated astrocytes and microglia, at 72 hpi. However, astrocytes and microglia were smaller after pre-traumatic microbiome depletion compared to vehicle treatment at 72 hpi, indicating less inflammatory activation. Accordingly, TBI-induced gene expression of the inflammation markers Interleukin-1ß, complement component C3, translocator protein TSPO and the major histocompatibility complex MHC2 was attenuated in microbiome-depleted mice along with reduced Immunoglobulin G extravasation as a proxy of blood-brain barrier (BBB) impairment. These results suggest that the gut microbiome contributes to early neuroinflammatory responses to TBI but does not have a significant impact on brain histopathology and neurological deficits. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

AT 1 inhibition mediated neuroprotection after experimental traumatic brain injury is dependent on neutrophils in male mice.

After traumatic brain injury (TBI) cerebral inflammation with invasion of neutrophils and lymphocytes is a crucial factor in the process of secondary brain damage. In TBI the intrinsic renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) reduces secondary brain damage and the invasion of neutrophil granulocytes into injured cerebral tissue. The current study explored the involvement of immune cells in neuroprotection mediated by AT1 inhibition following experimental TBI. Four different cohorts of male mice were examined, investigating the effects of neutropenia (anti-Ly6G antibody mediated neutrophil depletion; C57BL/6), lymphopenia (RAG1 deficiency, RAG1-/-), and their combination with candesartan-mediated AT1 inhibition. The present results showed that reduction of neutrophils and lymphocytes, as well as AT1 inhibition in wild type and RAG1-/- mice, reduced brain damage and neuroinflammation after TBI. However, in neutropenic mice, candesartan did not have an effect. Interestingly, AT1 inhibition was found to be neuroprotective in RAG1-/- mice but not in neutropenic mice. The findings suggest that AT1 inhibition may exert neuroprotection by reducing the inflammation caused by neutrophils, ultimately leading to a decrease in their invasion into cerebral tissue.

Inhibition of spinal ferroptosis-like cell death alleviates hyperalgesia and spontaneous pain in a mouse model of bone cancer pain.

Bone cancer pain (BCP) impairs patients' quality of life. However, the underlying mechanisms are still unclear. This study investigated the role of spinal interneuron death using a pharmacological inhibitor of ferroptosis in a mouse model of BCP. Lewis lung carcinoma cells were inoculated into the femur, resulting in hyperalgesia and spontaneous pain. Biochemical analysis revealed that spinal levels of reactive oxygen species and malondialdehyde were increased, while those of superoxide dismutase were decreased. Histological analysis showed the loss of spinal GAD65+ interneurons and provided ultrastructural evidence of mitochondrial shrinkage. Pharmacologic inhibition of ferroptosis using ferrostatin-1 (FER-1, 10 mg/kg, intraperitoneal for 20 consecutive days) attenuated ferroptosis-associated iron accumulation and lipid peroxidation and alleviated BCP. Furthermore, FER-1 inhibited the pain-associated activation of ERK1/2 and COX-2 expression and prevented the loss of GABAergic interneurons. Moreover, FER-1 improved analgesia by the COX-2 inhibitor Parecoxib. Taken together, this study shows that pharmacological inhibition of ferroptosis-like cell death of spinal interneurons alleviates BCP in mice. The results suggest that ferroptosis is a potential therapeutic target in patients suffering on BCP and possibly other types of pain.

Early DNase-I therapy delays secondary brain damage after traumatic brain injury in adult mice.

Traumatic brain injury (TBI) causes the release of danger-associated molecular patterns (DAMP) from damaged or dead cells, which contribute to secondary brain damage after TBI. Cell-free DNA (cfDNA) is a DAMP known to cause disruption of the blood-brain barrier (BBB), promote procoagulant processes, brain edema, and neuroinflammation. This study tested the hypothesis that administration of deoxyribonuclease-I (DNase-I) has a beneficial effect after TBI. Mice (n = 84) were subjected to controlled cortical impact (CCI) and posttraumatic intraperitoneal injections of low dose (LD) or high dose (HD) of DNase-I or vehicle solution at 30 min and 12 h after CCI. LD was most effective to reduce lesion volume (p = 0.003), brain water content (p < 0.0001) and to stabilize BBB integrity (p = 0.019) 1 day post-injury (dpi). At 6 h post injury LD-treated animals showed less cleavage of fibrin (p = 0.0014), and enhanced perfusion as assessed by micro-computer-tomography (p = 0.027). At 5 dpi the number of Iba1-positive cells (p = 0.037) were reduced, but the number of CD45-positive cells, motoric function and brain lesion volume was not different. Posttraumatic-treatment with DNase-I therefore stabilizes the BBB, reduces the formation of brain edema, immune response, and delays secondary brain damage. DNase-I might be a new approach to extend the treatment window after TBI.

Depression-Associated Negr1 Gene-Deficiency Induces Alterations in the Monoaminergic Neurotransmission Enhancing Time-Dependent Sensitization to Amphetamine in Male Mice.

In GWAS studies, the neural adhesion molecule encoding the neuronal growth regulator 1 (NEGR1) gene has been consistently linked with both depression and obesity. Although the linkage between NEGR1 and depression is the strongest, evidence also suggests the involvement of NEGR1 in a wide spectrum of psychiatric conditions. Here we show the expression of NEGR1 both in tyrosine- and tryptophan hydroxylase-positive cells. Negr1-/- mice show a time-dependent increase in behavioral sensitization to amphetamine associated with increased dopamine release in both the dorsal and ventral striatum. Upregulation of transcripts encoding dopamine and serotonin transporters and higher levels of several monoamines and their metabolites was evident in distinct brain areas of Negr1-/- mice. Chronic (23 days) escitalopram-induced reduction of serotonin and dopamine turnover is enhanced in Negr1-/- mice, and escitalopram rescued reduced weight of hippocampi in Negr1-/- mice. The current study is the first to show alterations in the brain monoaminergic systems in Negr1-deficient mice, suggesting that monoaminergic neural circuits contribute to both depressive and obesity-related phenotypes linked to the human NEGR1 gene.

Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Extracellular signal-regulated kinase-dependent phosphorylation of histone H3 serine 10 is involved in the pathogenesis of traumatic brain injury.

Traumatic brain injury (TBI) induces a series of epigenetic changes in brain tissue, among which histone modifications are associated with the deterioration of TBI. In this study, we explored the role of histone H3 modifications in a weight-drop model of TBI in rats. Screening for various histone modifications, immunoblot analyses revealed that the phosphorylation of histone H3 serine 10 (p-H3S10) was significantly upregulated after TBI in the brain tissue surrounding the injury site. A similar posttraumatic regulation was observed for phosphorylated extracellular signal-regulated kinase (p-ERK), which is known to phosphorylate H3S10. In support of the hypothesis that ERK-mediated phosphorylation of H3S10 contributes to TBI pathogenesis, double immunofluorescence staining of brain sections showed high levels and colocalization of p-H3S10 and p-ERK predominantly in neurons surrounding the injury site. To test the hypothesis that inhibition of ERK-H3S10 signaling ameliorates TBI pathogenesis, the mitogen-activated protein kinase-extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitor U0126, which inhibits ERK phosphorylation, was administered into the right lateral ventricle of TBI male and female rats via intracerebroventricular cannulation for 7 days post trauma. U0126 administration indeed prevented H3S10 phosphorylation and improved motor function recovery and cognitive function compared to vehicle treatment. In agreement with our findings in the rat model of TBI, immunoblot and double immunofluorescence analyses of brain tissue specimens from patients with TBI demonstrated high levels and colocalization of p-H3S10 and p-ERK as compared to control specimens from non-injured individuals. In conclusion, our findings indicate that phosphorylation-dependent activation of ERK-H3S10 signaling participates in the pathogenesis of TBI and can be targeted by pharmacological approaches.

Microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity.

Microglia are phagocytosis-competent CNS cells comprising a spectrum of subtypes with beneficial and/or detrimental functions in acute and chronic neurodegenerative disorders. The heterogeneity of microglia suggests differences in phagocytic activity and phenotype plasticity between microglia subtypes. To study these issues, primary murine glial cultures were cultivated in the presence of serum, different growth factors and cytokines to obtain M0-like, M1-like, and M2-like microglia as confirmed by morphology, M1/M2 gene marker expression, and nitric oxide assay. Single-cell analysis after 3 hours of phagocytosis of E.coli particles or IgG-opsonized beads showed equal internalization by M0-like microglia, whereas M1-like microglia preferably internalized E.coli particles and M2-like microglia preferably internalized IgG beads, suggesting subtype-specific preferences for different phagocytosis substrates. Time-lapse live-cells imaging over 16 hours revealed further differences between microglia subtypes in phagocytosis preference and internalization dynamics. M0- and, more efficiently, M1-like microglia continuously internalized E.coli particles for 16 hours, whereas M2-like microglia discontinued internalization after approximately 8 hours. IgG beads were continuously internalized by M0- and M1-like microglia but strikingly less by M2-like microglia. M2-like microglia initially showed continuous internalization similar to M0-like microglia but again discontinuation of internalization after 8 hours suggesting that the time of substrate exposure differently affect microglia subtypes. After prolonged exposure to E.coli particles or IgG beads for 5 days all microglia subtypes showed increased internalization of E.coli particles compared to IgG beads, increased nitric oxide release and up-regulation of M1 gene markers, irrespectively of the phagocytosis substrate, suggesting phenotype plasticity. In summary, microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity. The results suggest that prolonged phagocytosis substrate exposure enhances M1-like profiles and M2-M1 repolarization of microglia. Similar processes may also take place in conditions of acute and chronic brain insults when microglia encounter different types of phagocytic substrates.

Early posttraumatic CSF1R inhibition via PLX3397 leads to time- and sex-dependent effects on inflammation and neuronal maintenance after traumatic brain injury in mice.

BACKGROUND: There is a need for early therapeutic interventions after traumatic brain injury (TBI) to prevent neurodegeneration. Microglia/macrophage (M/M) depletion and repopulation after treatment with colony stimulating factor 1 receptor (CSF1R) inhibitors reduces neurodegeneration. The present study investigates short- and long-term consequences after CSF1R inhibition during the early phase after TBI. METHODS: Sex-matched mice were subjected to TBI and CSF1R inhibition by PLX3397 for 5 days and sacrificed at 5 or 30 days post injury (dpi). Neurological deficits were monitored and brain tissues were examined for histo- and molecular pathological markers. RNAseq was performed with 30 dpi TBI samples. RESULTS: At 5 dpi, CSF1R inhibition attenuated the TBI-induced perilesional M/M increase and associated gene expressions by up to 50%. M/M attenuation did not affect structural brain damage at this time-point, impaired hematoma clearance, and had no effect on IL-1ß expression. At 30 dpi, following drug discontinuation at 5 dpi and M/M repopulation, CSF1R inhibition attenuated brain tissue loss regardless of sex, as well as hippocampal atrophy and thalamic neuronal loss in male mice. Selected gene markers of brain inflammation and apoptosis were reduced in males but increased in females after early CSF1R inhibition as compared to corresponding TBI vehicle groups. Neurological outcome in behaving mice was almost not affected. RNAseq and gene set enrichment analysis (GSEA) of injured brains at 30 dpi revealed more genes associated with dendritic spines and synapse function after early CSF1R inhibition as compared to vehicle, suggesting improved neuronal maintenance and recovery. In TBI vehicle mice, GSEA showed high oxidative phosphorylation, oxidoreductase activity and ribosomal biogenesis suggesting oxidative stress and increased abundance of metabolically highly active cells. More genes associated with immune processes and phagocytosis in PLX3397 treated females vs males, suggesting sex-specific differences in response to early CSF1R inhibition after TBI. CONCLUSIONS: M/M attenuation after CSF1R inhibition via PLX3397 during the early phase of TBI reduces long-term brain tissue loss, improves neuronal maintenance and fosters synapse recovery. Overall effects were not sex-specific but there is evidence that male mice benefit more than female mice.

Cerebral Tissue Oxygen Saturation Correlates with Emergence from Propofol-Remifentanil Anesthesia: An Observational Cohort Study.

Anesthesia emergence is accompanied by changes in cerebral circulation. It is unknown whether cerebral tissue oxygen saturation (SctO2) could be an indicator of emergence. Changes in SctO2, bispectral index (BIS), mean arterial pressure (MAP), and heart rate (HR) were evaluated during the emergence from propofol-remifentanil anesthesia. At the time of cessation of anesthetic delivery, SctO2, BIS, MAP, and HR values were recorded as baseline. The changes of these parameters from the baseline were recorded as Δ SctO2, Δ BIS, Δ MAP, and Δ HR. The behavioral signs (body movement, coughing, or eye opening) and response to commands (indicating regaining of consciousness) were used to define emergence states. Prediction probability (Pk) was used to examine the accuracy of SctO2, BIS, MAP, and HR as indicators of emergence. SctO2 showed an abrupt and distinctive increase when appearing behavioral signs. BIS, MAP, and HR, also increased but with a large inter-individual variability. Pk value of Δ SctO2 was 0.97 to predict the appearance behavioral signs from 2 min before that, which was much higher than the Pk values of Δ BIS (0.81), Δ MAP (0.71) and Δ HR (0.87). The regaining of consciousness was associated with a further increase in the SctO2 value.