Fingolimod protects against neonatal white matter damage and long-term cognitive deficits caused by hyperoxia.

Cerebral white matter injury is a leading cause of adverse neurodevelopmental outcome in prematurely born infants involving cognitive deficits in later life. Despite increasing knowledge about the pathophysiology of perinatal brain injury, therapeutic options are limited. In the adult demyelinating disease multiple sclerosis the sphingosine-1-phosphate (S1P) receptor modulating substance fingolimod (FTY720) has beneficial effects. Herein, we evaluated the neuroprotective potential of FTY720 in a neonatal model of oxygen-toxicity, which is associated with hypomyelination and impaired neuro-cognitive outcome. A single dose of FTY720 (1mg/kg) at the onset of neonatal hyperoxia (24h 80% oxygen on postnatal day 6) resulted in improvement of neuro-cognitive development persisting into adulthood. This was associated with reduced microstructural white matter abnormalities 4 months after the insult. In search of the underlying mechanisms potential non-classical (i.e. lymphocyte-independent) pathways were analysed shortly after the insult, comprising modulation of oxidative stress and local inflammatory responses as well as myelination, oligodendrocyte degeneration and maturation. Treatment with FTY720 reduced hyperoxia-induced oxidative stress, microglia activation and associated pro-inflammatory cytokine expression. In vivo and in vitro analyses further revealed that oxygen-induced hypomyelination is restored to control levels, which was accompanied by reduced oligodendrocyte degeneration and enhanced maturation. Furthermore, hyperoxia-induced elevation of S1P receptor 1 (S1P1) protein expression on in vitro cultured oligodendrocyte precursor cells was reduced by activated FTY720 and protection from degeneration is abrogated after selective S1P1 blockade. Finally, FTY720s' classical mode of action (i.e. retention of immune cells within peripheral lymphoid organs) was analysed demonstrating that FTY720 diminished circulating lymphocyte counts independent from hyperoxia. Cerebral immune cell counts remained unchanged by hyperoxia and by FTY720 treatment. Taken together, these results suggest that beneficial effects of FTY720 in neonatal oxygen-induced brain injury may be rather attributed to its anti-oxidative and anti-inflammatory capacity acting in concert with a direct protection of developing oligodendrocytes than to a modulation of peripheral lymphocyte trafficking. Thus, FTY720 might be a potential new therapeutic option for the treatment of neonatal brain injury through reduction of white matter damage.

Involvement of CXCL1/CXCR2 During Microglia Activation Following Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Microglia are key mediators of inflammation during perinatal brain injury. As shown experimentally after inflammation-sensitized hypoxic ischemic (HI) brain injury, microglia are activated into a pro-inflammatory status 24 h after HI involving the NLRP3 inflammasome pathway. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been shown to be involved in NLRP3 activation, although their specific role during perinatal brain injury remains unclear. In this study we investigated the involvement of CXCL1/CXCR2 in brain tissue and microglia and brain tissue after inflammation-sensitized HI brain injury of newborn rats. Methods: Seven-day old Wistar rat pups were either injected with vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia (8% O2 for 50 min). Pups were randomized into four different treatment groups: (1) Sham group (n = 21), (2) LPS only group (n = 20), (3) Veh/HI group (n = 39), and (4) LPS/HI group (n = 42). Twenty-four hours post hypoxia transcriptome and gene expression analysis were performed on ex vivo isolated microglia cells in our model. Additionally protein expression was analyzed in different brain regions at the same time point. Results: Transcriptome analyses showed a significant microglial upregulation of the chemokine CXCL1 and its receptor CXCR2 in the LPS/HI group compared with the other groups. Gene expression analysis showed a significant upregulation of CXCL1 and NLRP3 in microglia cells after inflammation-sensitized hypoxic-ischemic brain injury. Additionally, protein expression of CXCL1 was significantly upregulated in cortex of male pups from the LPS/HI group. Conclusion: These results indicate that the CXCL1/CXCR2 pathway may be involved during pro-inflammatory microglia activation following inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats. This may lead to new treatment options altering CXCR2 activation early after HI brain injury.

Detrimental Impact of Energy Drink Compounds on Developing Oligodendrocytes and Neurons.

The consumption of energy drinks is continuously rising, particularly in children and adolescents. While risks for adverse health effects, like arrhythmia, have been described, effects on neural cells remain elusive. Considering that neurodevelopmental processes like myelination and neuronal network formation peak in childhood and adolescence we hypothesized that developing oligodendrocytes and neurons are particularly vulnerable to main energy drink components. Immature oligodendrocytes and hippocampal neurons were isolated from P0-P1 Wistar rats and were incubated with 0.3 mg/mL caffeine and 4 mg/mL taurine alone or in combination for 24 h. Analysis was performed immediately after treatment or after additional three days under differentiating conditions for oligodendrocytes and standard culture for neurons. Oligodendrocyte degeneration, proliferation, and differentiation were assessed via immunocytochemistry and immunoblotting. Neuronal integrity was investigated following immunocytochemistry by analysis of dendrite outgrowth and axonal morphology. Caffeine and taurine induced an increased degeneration and inhibited proliferation of immature oligodendrocytes accompanied by a decreased differentiation capacity. Moreover, dendritic branching and axonal integrity of hippocampal neurons were negatively affected by caffeine and taurine treatment. The negative impact of caffeine and taurine on developing oligodendrocytes and disturbed neuronal morphology indicates a high risk for disturbed neurodevelopment in children and adolescents by excessive energy drink consumption.

Early Pro-inflammatory Microglia Activation After Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. Therapeutic hypothermia is only neuroprotective in 50% of cooled asphyxiated newborns. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. Microglia are thought to be key players after inflammation-sensitized HI brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes. Methods: Seven days old Wistar rat pups were injected with either vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia inducing a mild unilateral hypoxic-ischemic injury. Pups were randomized to (1) Sham group (n = 41), (2) LPS only group (n = 37), (3) Veh/HI group (n = 56), and (4) LPS/HI group (n = 79). On postnatal days 8 and 14 gene-expression analysis or immunohistochemistry was performed describing early microglia polarization in our model. Results: We confirmed that LPS pre-sensitization significantly increases brain area loss and induced microglia activation and neuronal injury after mild hypoxia-ischemia. Additionally, we show that microglia upregulate pro-inflammatory genes involving NLRP-3 inflammasome gene expression 24 h after inflammation-sensitized hypoxic-ischemic brain injury. Conclusion: These results demonstrate that microglia are early key mediators of the inflammatory response following inflammation-sensitized HI brain injury and that they polarize into a predominant pro-inflammatory phenotype 24 h post HI. This may lead to new treatment options altering microglia phenotype polarization early after HI brain injury.

Protection of Oligodendrocytes Through Neuronal Overexpression of the Small GTPase Ras in Hyperoxia-Induced Neonatal Brain Injury.

Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.

Heat shock protein 60: identification of specific epitopes for binding to primary macrophages.

In the present study, we characterized regions of human heat shock protein (HSP) 60 responsible for binding to primary macrophages. Studies using 20-mer peptides of the HSP60 sequence to compete with HSP60-binding to macrophages from C57BL/6J mice showed that regions aa241-260, aa391-410 and aa461-480 are involved in surface-binding. HSP60 mutants, lacking the N-terminal 137, 243 or 359 amino acids, inhibited HSP60-binding to primary macrophages to different degrees, demonstrating that all three regions are required for optimal binding. Analysis of different pro- and eukaryotic HSP60 species indicated that phylogenetically separate HSP60 species use different binding sites on primary macrophages.

Erythropoietin Restores Long-Term Neurocognitive Function Involving Mechanisms of Neuronal Plasticity in a Model of Hyperoxia-Induced Preterm Brain Injury.

Cerebral white and grey matter injury is the leading cause of an adverse neurodevelopmental outcome in prematurely born infants. High oxygen concentrations have been shown to contribute to the pathogenesis of neonatal brain damage. Here, we focused on motor-cognitive outcome up to the adolescent and adult age in an experimental model of preterm brain injury. In search of the putative mechanisms of action we evaluated oligodendrocyte degeneration, myelination, and modulation of synaptic plasticity-related molecules. A single dose of erythropoietin (20,000 IU/kg) at the onset of hyperoxia (24 hours, 80% oxygen) in 6-day-old Wistar rats improved long-lasting neurocognitive development up to the adolescent and adult stage. Analysis of white matter structures revealed a reduction of acute oligodendrocyte degeneration. However, erythropoietin did not influence hypomyelination occurring a few days after injury or long-term microstructural white matter abnormalities detected in adult animals. Erythropoietin administration reverted hyperoxia-induced reduction of neuronal plasticity-related mRNA expression up to four months after injury. Thus, our findings highlight the importance of erythropoietin as a neuroregenerative treatment option in neonatal brain injury, leading to improved memory function in adolescent and adult rats which may be linked to increased neuronal network connectivity.

Identification of the heat shock protein 60 epitope involved in receptor binding on macrophages.

In the present study, we identified the human heat shock protein 60 (HSP60) epitope responsible for binding to macrophages. Studies using overlapping 15- and 20-mer peptides of the human HSP60 sequence to compete with binding of HSP60 to macrophages indicated that surface binding was accounted for by the region aa481-500. Deletion mutants of HSP60, lacking the N-terminal 137, 243 or 359 amino acids, strongly inhibited HSP60 binding to macrophages. Monoclonal antibodies addressing regions aa1-200, aa335-366 or aa383-447 did not block HSP60 binding. We conclude that a single C-terminal region, aa481-500, accounts for the binding of HSP60 to macrophages.

Different heat shock protein 60 species share pro-inflammatory activity but not binding sites on macrophages.

In a study of seven different hsp60 species, we found that all mammalian and microbial proteins shared the property of eliciting an inflammatory response in mouse macrophages. In all cases, TNFalpha production was induced by 0.1 microM concentrations of hsp60. However, the different hsp60 preparations did not compete for the same binding site. The binding of fluorescence-labeled human hsp60 was inhibited by excess unlabeled human, rat or mouse hsp60, but not hamster, Escherichia coli, Chlamydia pneumoniae or Mycobacterium bovis hsp60. We conclude that phylogenetically separate hsp60 species interact with innate immune cells via different recognition pathways.

Heat shock protein 60: specific binding of lipopolysaccharide.

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.