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.

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.

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

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.

Posttraumatic midazolam administration does not influence brain damage after experimental traumatic brain injury.

BACKGROUND: The benzodiazepine midazolam is a γ-aminobutyric acid (GABA)-A receptor agonist frequently used for sedation or stress control in patients suffering from traumatic brain injury (TBI). However, experimental studies on benzodiazepines have reported divergent results, raising concerns about its widespread use in patients. Some studies indicate that benzodiazepine-mediated potentiation of GABAergic neurotransmission is detrimental in brain-injured animals. However, other experimental investigations demonstrate neuroprotective effects, especially in pretreatment paradigms. This study investigated whether single-bolus midazolam administration influences secondary brain damage post-TBI. METHODS: Two different midazolam dosages (0.5 and 5 mg/kg BW), a combination of midazolam and its competitive antagonist flumazenil, or vehicle solution (NaCl 0.9%) was injected intravenously to mice 24 h after experimental TBI induced by controlled cortical impact. Mice were evaluated for neurological and motor deficits using a 15-point neuroscore and the rotarod test. Histopathological brain damage and mRNA expression of inflammatory marker genes were analyzed using quantitative polymerase chain reaction three days after insult. RESULTS: Histological brain damage was not affected by posttraumatic midazolam administration. Midazolam impaired functional recovery, and this effect could not be counteracted by administering the midazolam antagonist flumazenil. An increase in IL-1ß mRNA levels due to postinjury application of midazolam was reversible by flumazenil administration. However, other inflammatory parameters were not affected. CONCLUSIONS: This study merely reports minor effects of a postinjury midazolam application. Further studies focusing on a time-dependent analysis of posttraumatic benzodiazepine administration are required.

Single intracerebroventricular progranulin injection adversely affects the blood-brain barrier in experimental traumatic brain injury.

Progranulin (PGRN) is a neurotrophic and anti-inflammatory factor with protective effects in animal models of ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury (TBI). Administration of recombinant (r) PGRN prevents exaggerated brain pathology after TBI in Grn-deficient mice, suggesting that local injection of recombinant progranulin (rPGRN) provides therapeutic benefit in the acute phase of TBI. To test this hypothesis, we subjected adult male C57Bl/6N mice to the controlled cortical impact model of TBI, administered a single dose of rPGRN intracerebroventricularly (ICV) shortly before the injury, and examined behavioral and biological effects up to 5 days post injury (dpi). The anti-inflammatory bioactivity of rPGRN was confirmed by its capability to inhibit the inflammation-induced hypertrophy of murine primary microglia and astrocytes in vitro. In C57Bl/6N mice, however, ICV administration of rPGRN failed to attenuate behavioral deficits over the 5-day observation period. (Immuno)histological gene and protein expression analyses at 5 dpi did not reveal a therapeutic benefit in terms of brain injury size, brain inflammation, glia activation, cell numbers in neurogenic niches, and neuronal damage. Instead, we observed a failure of TBI-induced mRNA upregulation of the tight junction protein occludin and increased extravasation of serum immunoglobulin G into the brain parenchyma at 5 dpi. In conclusion, single ICV administration of rPGRN had not the expected protective effects in the acute phase of murine TBI, but appeared to cause an aggravation of blood-brain barrier disruption. The data raise questions about putative PGRN-boosting approaches in other types of brain injuries and disease.

Early Reciprocal Effects in a Murine Model of Traumatic Brain Injury and Femoral Fracture.

Traumatic brain injury (TBI) represents a major cause of death and disability in early adulthood. Concomitant extracranial injury such as long bone fracture was reported to exacerbate TBI pathology. However, early reciprocal effects and mechanisms have been barely investigated. To address this issue, C57BL/6N mice were subjected to either the controlled cortical impact (CCI) model of TBI, fracture of the left femur (FF), combined injury (CCI+FF), or sham procedure. Behavioral alterations were monitored until 5 days post injury (dpi), followed by (immuno-)histology, gene and protein expression analyses using quantitative PCR, western blot, and ELISA. We found that CCI+FF mice exhibited increased neurological impairments, reduced recovery, and altered anxiety-related behavior compared to single injury groups. At 5 dpi, cerebral lesion size was not affected by combined injury but exaggerated hippocampal substance loss and increased perilesional astrogliosis were observed in CCI+FF mice compared to isolated CCI. Bone gene expression of the osteogenic markers Runx2, osteocalcin, alkaline phosphatase, and bone sialoprotein was induced by fracture injury but attenuated by concomitant TBI. Plasma concentrations of the biomarkers osteopontin and progranulin were elevated in CCI+FF mice compared to other experimental groups. Taken together, using a murine model of TBI and femoral fracture, we report early reciprocal impairments of brain tissue maintenance, behavioral recovery, and bone repair gene expression. Increased circulating levels of the biomarkers osteopontin and progranulin indicate ongoing tissue inflammation and repair. Our results may have implications for future therapeutic approaches to interfere with the pathological crosstalk between TBI and concomitant bone fracture.

Effect of fluid resuscitation on cerebral integrity: A prospective randomised porcine study of haemorrhagic shock.

BACKGROUND: The treatment of haemorrhagic shock is a challenging task. Colloids have been regarded as standard treatment, but their safety and benefit have been the subject of controversial debates. Negative effects, including renal failure and increased mortality, have resulted in restrictions on their administration. The cerebral effects of different infusion regimens are largely unknown. OBJECTIVES: The current study investigated the impact of gelatine-polysuccinate, hydroxyethyl starch (HES) and balanced electrolyte solution (BES) on cerebral integrity, focusing on cerebral inflammation, apoptosis and blood flow in pigs. DESIGN: Randomised experimental study. SETTING: University-affiliated large animal research unit. ANIMALS: Twenty-four juvenile pigs aged 8 to 12 weeks. INTERVENTION: Haemorrhagic shock was induced by controlled arterial blood withdrawal to achieve a combination of relevant blood loss (30 to 40 ml kg-1) and haemodynamic deterioration. After 30 min of shock, fluid resuscitation was started with either gelatine-polysuccinate, HES or BES. The animals were then monitored for 4 h. MAIN OUTCOME MEASURES: Cerebral perfusion and diffusion were measured via arterial-spin-labelling MRI. Peripheral tissue perfusion was evaluated via white light spectroscopy. Cortical and hippocampal samples were collected at the end of the experiment. The numbers of cerebral cell nuclei were counted and mRNA expression of markers for cerebral apoptosis [glucose transporter protein type 1 (SLC2A), lipocalin 2 (LCN-2), aquaporin-4 (AQP4)] and inflammation [IL-6, TNF-α, glial fibrillary acidic protein (GFAP)] were determined. RESULTS: The three fluid protocols all stabilised the macrocirculation. Fluid resuscitation significantly increased the cerebral perfusion. Gelatine-polysuccinate and HES initially led to a higher cardiac output but caused haemodilution. Cerebral cell counts (as cells µm-2) were lower after colloid administration in the cortex (gelatine-polysuccinate, 1.8 ±â€Š0.3; HES, 1.9 ±â€Š0.4; each P < 0.05 vs. BES, 2.3 ±â€Š0.2) and the hippocampus (gelatine-polysuccinate, 0.8 ±â€Š0.2; HES, 0.9 ±â€Š0.2; each P < 0.05 vs. BES, 1.1 ±â€Š0.1). After gelatine-polysuccinate, the hippocampal SLC2A and GFAP were lower. After gelatine-polysuccinate, the cortical LCN-2 and TNF-α expression levels were increased (each P < 0.05 vs. BES). CONCLUSION: In a porcine model, fluid resuscitation by colloids, particularly gelatine-polysuccinate, was associated with the occurrence of cerebral injury. ETHICAL APPROVAL NUMBER: 23 177-07/G 15-1-092; 01/2016.

Targeting aurora kinase B alleviates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury.

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury.

Sex hormones modulate pathogenic processes in experimental traumatic brain injury.

Clinical and animal studies have revealed sex-specific differences in histopathological and neurological outcome after traumatic brain injury (TBI). The impact of perioperative administration of sex steroid inhibitors on TBI is still elusive. Here, we subjected male and female C57Bl/6N mice to the controlled cortical impact (CCI) model of TBI and applied pharmacological inhibitors of steroid hormone synthesis, that is, letrozole (LET, inhibiting estradiol synthesis by aromatase) and finasteride (FIN, inhibiting dihydrotestosterone synthesis by 5α-reductase), respectively, starting 72 h prior CCI, and continuing for a further 48 h after CCI. Initial gene expression analyses showed that androgen (Ar) and estrogen receptors (Esr1) were sex-specifically altered 72 h after CCI. When examining brain lesion size, we found larger lesions in male than in female mice, but did not observe effects of FIN or LET treatment. However, LET treatment exacerbated neurological deficits 24 and 72 h after CCI. On the molecular level, FIN administration reduced calpain-dependent spectrin breakdown products, a proxy of excitotoxicity and disturbed Ca2+ homeostasis, specifically in males, whereas LET increased the reactive astrocyte marker glial fibrillary acid protein specifically in females. Examination of neurotrophins (brain-derived neurotrophic factor, neuronal growth factor, NT-3) and their receptors (p75NTR , TrkA, TrkB, TrkC) revealed CCI-induced down-regulation of TrkB and TrkC protein expression, which was reduced by LET in both sexes. Interestingly, FIN decreased neuronal growth factor mRNA expression and protein levels of its receptor TrkA only in males. Taken together, our data suggest a sex-specific impact on pathogenic processes in the injured brain after TBI. Sex hormones may thus modulate pathogenic processes in experimental TBI.

Correction to: Depletion of regulatory T cells increases T cell brain infiltration, reactive astrogliosis, and interferon-γ gene expression in acute experimental traumatic brain injury.

Following publication of the original article [1], the authors opted to correct the following mistakes. According to the title and our results, the conclusions in the abstract and at the end of the discussion the term "attenuates" must be corrected to read as "increases".

Depletion of regulatory T cells increases T cell brain infiltration, reactive astrogliosis, and interferon-γ gene expression in acute experimental traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. T cells were shown to infiltrate the brain during the first days after injury and to exacerbate tissue damage. The objective of this study was to investigate the hitherto unresolved role of immunosuppressive, regulatory T cells (Tregs) in experimental TBI. METHODS: "Depletion of regulatory T cell" (DEREG) and wild type (WT) C57Bl/6 mice, treated with diphtheria toxin (DTx) to deplete Tregs or to serve as control, were subjected to the controlled cortical impact (CCI) model of TBI. Neurological and motor deficits were examined until 5 days post-injury (dpi). At the 5 dpi endpoint, (immuno-) histological, protein, and gene expression analyses were carried out to evaluate the consequences of Tregs depletion. Comparison of parametric or non-parametric data between two groups was done using Student's t test or the Mann-Whitney U test. For multiple comparisons, p values were calculated by one-way or two-way ANOVA followed by specific post hoc tests. RESULTS: The overall neurological outcome at 5 dpi was not different between DEREG and WT mice but more severe motor deficits occurred transiently at 1 dpi in DEREG mice. DEREG and WT mice did not differ in the extent of brain damage, blood-brain barrier (BBB) disruption, or neuronal excitotoxicity, as examined by lesion volumetry, immunoglobulin G (IgG) extravasation, or calpain-generated αII-spectrin breakdown products (SBDPs), respectively. In contrast, increased protein levels of glial fibrillary acidic protein (GFAP) and GFAP+ astrocytes in the ipsilesional brain tissue indicated exaggerated reactive astrogliosis in DEREG mice. T cell counts following anti-CD3 immunohistochemistry and gene expression analyses of Cd247 (CD3 subunit zeta) and Cd8a (CD8a) further indicated an increased number of T cells infiltrating the brain injury sites of DEREG mice compared to WT. These changes coincided with increased gene expression of pro-inflammatory interferon-γ (Ifng) in DEREG mice compared to WT in the injured brain. CONCLUSIONS: The results show that the depletion of Tregs attenuates T cell brain infiltration, reactive astrogliosis, interferon-γ gene expression, and transiently motor deficits in murine acute traumatic brain injury.

SETD7 mediates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury.

Gene transcription regulation is critical for the development of spinal microgliosis and neuropathic pain after peripheral nerve injury. Using a model of chronic constriction injury (CCI) of the sciatic nerve, this study characterized the role of SET domain containing lysine methyltransferase 7 (SETD7) which monomethylates histone H3 lysine 4 (H3K4me1), a marker for active gene transcription. SETD7 protein expression in the spinal dorsal horn ipsilateral to nerve lesion was increased from one day to 14 days after CCI, concomitantly with the expression of inflammatory genes, Ccl2, Il-6 and Il-1ß. The CCI-induced SETD7 expression was predominantly localized to microglia, as demonstrated by immunohistochemistry and western blot from magnetic activated cell sorted spinal microglia. SETD7 knockdown by intrathecal lentivirus shRNA delivery prior to CCI prevented spinal microgliosis and neuropathic pain, whereas lentiviral SETD7 transduction exacerbated these symptoms. In addition, SETD7 regulated H3K4me1 level and expression of inflammatory mediators both in CCI rats and in the HAPI rat microglia cell line. Accordingly, PFI-2, a specific inhibitor of SETD7 monomethylation activity, suppressed the lipopolysaccharides-induced amoeboid morphology of primary microglia and the expression of inflammatory genes, Ccl2, Il-6 and Il-1ß. Moreover, intrathecal administration of PFI-2 alleviated CCI-induced neuropathic pain. However, this effect was observed in male but not in female rats. These results demonstrate a critical role of SETD7 in the development of spinal microgliosis and neuropathic pain subsequently to peripheral nerve injury. The pharmacological approach further suggests that SETD7 is a new target for the treatment of neuropathic pain. The underlying mechanisms may involve H3K4me1-dependent regulation of inflammatory gene expression in microglia.

Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala.

The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17ß-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17ß-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.

Progranulin protects against exaggerated axonal injury and astrogliosis following traumatic brain injury.

In response to traumatic brain injury (TBI) microglia/macrophages and astrocytes release inflammatory mediators with dual effects on secondary brain damage progression. The neurotrophic and anti-inflammatory glycoprotein progranulin (PGRN) attenuates neuronal damage and microglia/macrophage activation in brain injury but mechanisms are still elusive. Here, we studied histopathology, neurology and gene expression of inflammatory markers in PGRN-deficient mice (Grn-/- ) 24 h and 5 days after experimental TBI. Grn-/- mice displayed increased perilesional axonal injury even though the overall brain tissue loss and neurological consequences were similar to wild-type mice. Brain inflammation was elevated in Grn-/- mice as reflected by increased transcription of pro-inflammatory cytokines TNFα, IL-1ß, IL-6, and decreased transcription of the anti-inflammatory cytokine IL-10. However, numbers of Iba1+ microglia/macrophages and immigrated CD45+ leukocytes were similar at perilesional sites while determination of IgG extravasation suggested stronger impairment of blood brain barrier integrity in Grn-/- compared to wild-type mice. Most strikingly, Grn-/- mice displayed exaggerated astrogliosis 5 days after TBI as demonstrated by anti-GFAP immunohistochemistry and immunoblot. GFAP+ astrocytes at perilesional sites were immunolabelled for iNOS and TNFα suggesting that pro-inflammatory activation of astrocytes was attenuated by PGRN. Accordingly, recombinant PGRN (rPGRN) attenuated LPS- and cytokine-evoked iNOS and TNFα mRNA expression in cultured astrocytes. Moreover, intracerebroventricular administration of rPGRN immediately before trauma reduced brain damage and neurological deficits, and restored normal levels of cytokine transcription, axonal injury and astrogliosis 5 days after TBI in Grn-/- mice. Our results show that endogenous and recombinant PGRN limit axonal injury and astrogliosis and suggest therapeutic potential of PGRN in TBI. GLIA 2017;65:278-292.

Loss of synaptic zinc transport in progranulin deficient mice may contribute to progranulin-associated psychopathology and chronic pain.

Affective and cognitive processing of nociception contributes to the development of chronic pain and vice versa, pain may precipitate psychopathologic symptoms. We hypothesized a higher risk for the latter with immanent neurologic diseases and studied this potential interrelationship in progranulin-deficient mice, which are a model for frontotemporal dementia, a disease dominated by behavioral abnormalities in humans. Young naïve progranulin deficient mice behaved normal in tests of short-term memory, anxiety, depression and nociception, but after peripheral nerve injury, they showed attention-deficit and depression-like behavior, over-activity, loss of shelter-seeking, reduced impulse control and compulsive feeding behavior, which did not occur in equally injured controls. Hence, only the interaction of 'pain x progranulin deficiency' resulted in the complex phenotype at young age, but neither pain nor progranulin deficiency alone. A deep proteome analysis of the prefrontal cortex and olfactory bulb revealed progranulin-dependent alterations of proteins involved in synaptic transport, including neurotransmitter transporters of the solute carrier superfamily. In particular, progranulin deficiency was associated with a deficiency of nuclear and synaptic zinc transporters (ZnT9/Slc30a9; ZnT3/Slc30a3) with low plasma zinc. Dietary zinc supplementation partly normalized the attention deficit of progranulin-deficient mice, which was in part reminiscent of autism-like and compulsive behavior of synaptic zinc transporter Znt3-knockout mice. Hence, the molecular studies point to defective zinc transport possibly contributing to progranulin-deficiency-associated psychopathology. Translated to humans, our data suggest that neuropathic pain may precipitate cognitive and psychopathological symptoms of an inherent, still silent neurodegenerative disease.

Lack of NG2 exacerbates neurological outcome and modulates glial responses after traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2-knockout mice (NG2-KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2-KO compared to wild-type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin-2) were increased in the perilesional brain tissue of NG2-KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2-KO mice. While TBI-induced expression of pro-inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI-induced up-regulation of the C-X-C motif chemokine 13 gene Cxcl13 in NG2-KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2-KO mice still exhibited more pronounced neurological deficits than WT mice, up-regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba-1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI.

Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury.

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35-55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139-151-induced relapsing-remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury.

2-Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF-1α response after traumatic brain injury in mice.

HIF-1α is pivotal for cellular homeostasis in response to cerebral ischemia. Pharmacological inhibition of HIF-1α may reduce secondary brain damage by targeting post-translational mechanisms associated with its proteasomal degradation and nuclear translocation. This study examined the neuroprotective effects of 2-methoxyestradiol (2ME2), the involved HIF-1α-dependent response, and alternative splicing in exon 14 of HIF-1α (HIF-1α∆Ex14) after traumatic brain injury (TBI) in mice. Intraperitoneal 2ME2 administration 30 min after TBI caused a dose-dependent reduction in secondary brain damage after 24 h. 2ME2 was physiologically tolerated, showed no effects on immune cell brain migration, and mitigated trauma-induced brain expression of neuropathologically relevant HIF-1α target genes encoding for Plasminogen activator inhibitor 1 and tumor necrosis factor alpha. Moreover, TBI-induced expression of pro-apoptotic BNIP3 was attenuated by 2ME2 treatment. Alternatively, spliced HIF-1α∆Ex14 was substantially up-regulated from 6 to 48 h after TBI. In vitro, nuclear location and gene transcription activity of HIF-1α∆Ex14 were impaired compared to full-length HIF-1α, but no effects on nuclear translocation of the transcriptional complex partner HIF-1ß were observed. This study demonstrates that 2ME2 confers neuroprotection after TBI. While the role of alternatively spliced HIF-1α∆Ex14 remains elusive, the in vivo data provide evidence that inhibition of a maladaptive HIF-1α-dependent response contributes to the neuroprotective effects of 2ME2. We examined neuroprotective effects of 2-methoxyestradiol (2ME2) and the hypoxia-inducible factor 1-α (HIF-1α) response following traumatic brain injury in mice. Early 2ME2 administration reduced the secondary brain damage and neuronal HIF-1α probably involving ubiquitin proteasome system-mediated degradation. The up-regulation of neuropathological HIF-1α target genes and pro-apoptotic BNIP3 protein was attenuated. We propose that the inhibition of a maladaptive HIF-1α response may contribute to 2ME2-mediated neuroprotection.

Regulators of mitochondrial Ca(2+) homeostasis in cerebral ischemia.

Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca(2+) and is fueled either by Ca(2+) from the extracellular space when triggered by neuronal activity or by Ca(2+) released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca(2+) pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca(2+) homeostasis in models of ischemia. The proteins include those important for the Ca(2+)-dependent motility of mitochondria and for Ca(2+) transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca(2+) release from the ER, voltage-dependent anion channels that allow Ca(2+) entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca(2+) uniporter together with its regulatory proteins that permit Ca(2+) entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca(2+) from the mitochondria such as the mitochondrial Na(+)/Ca(2+) exchanger or, if the mitochondrial Ca(2+) concentration exceeds a certain threshold, the mitochondrial permeability transition pore.