TWEAK Receptor Deficiency Has Opposite Effects on Female and Male Mice Subjected to Neonatal Hypoxia-Ischemia.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine member of the TNF family. TWEAK binds to its only known receptor, Fn14, enabling it to activate downstream signaling processes in response to tissue injury. The aim of this study was to investigate the role of TWEAK signaling in neonatal hypoxia-ischemia (HI). We found that after neonatal HI, both TWEAK and Fn14 expression were increased to a greater extent in male compared with female mice. To assess the role of TWEAK signaling after HI, the size of the injury was measured in neonatal mice genetically deficient in Fn14 and compared with their wild-type and heterozygote littermates. A significant sex difference in the Fn14 knockout (KO) animals was observed. Fn14 gene KO was beneficial in females; conversely, reducing Fn14 expression exacerbated the brain injury in male mice. Our findings indicate that the TWEAK/Fn14 pathway is critical for development of hypoxic-ischemic brain injury in immature animals. However, as the responses are different in males and females, clinical implementation depends on development of sex-specific therapies.

Oxidative stress and endoplasmic reticulum (ER) stress in the development of neonatal hypoxic-ischaemic brain injury.

Birth asphyxia in term neonates affects 1-2/1000 live births and results in the development of hypoxic-ischaemic encephalopathy with devastating life-long consequences. The majority of neuronal cell death occurs with a delay, providing the potential of a treatment window within which to act. Currently, treatment options are limited to therapeutic hypothermia which is not universally successful. To identify new interventions, we need to understand the molecular mechanisms underlying the injury. Here, we provide an overview of the contribution of both oxidative stress and endoplasmic reticulum stress in the development of neonatal brain injury and identify current preclinical therapeutic strategies.

Implicating Receptor Activator of NF-κB (RANK)/RANK Ligand Signalling in Microglial Responses to Toll-Like Receptor Stimuli.

Inflammation in the perinatal brain caused by maternal or intrauterine fetal infection is now well established as an important contributor to the development of perinatal brain injury. Exposure to inflammatory products can impair perinatal brain development and act as a risk factor for neurological dysfunction, cognitive disorders, cerebral palsy, or preterm birth. Pre-exposure to inflammation significantly exacerbates brain injury caused by hypoxic/ischaemic insult. Tumour necrosis factor (TNF) is a family of cytokines largely involved in inflammation signalling. In our previous study, we identified the importance of TNF-related apoptosis-inducing ligand (TRAIL) signalling in the development of perinatal brain injury. We observed a significant increase in the expression levels of a soluble decoy receptor for TRAIL, osteoprotegerin (OPG). Besides TRAIL, OPG is able to bind the receptor activator of the NF-κB (RANK) ligand (RANKL) and inhibit its signalling. The function of the RANK/RANKL/OPG system in the brain has not come under much scrutiny. The aim of this research study was to elucidate the role of RANK, RANKL, and OPG in microglial responses to the proinflammatory stimuli lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C). Here, we show that RANK signalling is important for regulating the activation of the BV2 microglial cell line. We found that LPS treatment causes a significant decrease in the expression of RANK in the BV2 cell line while significantly increasing the expression of OPG, Toll-like receptor (TLR)3, and the adaptor proteins MyD88 and TRIF. We found that pretreatment of BV2 cells with RANKL for 24 h before the LPS or Poly I:C exposure decreases the expression of inflammatory markers such as inducible nitric oxide synthase and cyclooxygenase. This is accompanied by a decreased expression of the TLR adaptor proteins MyD88 and TRIF, which we observed after RANKL treatment. Similar results were obtained in our experiments with primary mouse microglia. Using recently developed CRISPR/Cas9 technology, we generated a BV2 cell line lacking RANK (RANK-/- BV2). We showed that most effects of RANKL pretreatment were abolished, thereby proving the specificity of this effect. Taken together, these findings suggest that RANK signalling is important for modulating the inflammatory activation of microglial cells to a moderate level, and that RANK attenuates TLR3/TLR4 signalling.

Inflammation-induced sensitization of the brain in term infants.

Perinatal insults are a leading cause of infant mortality and amongst survivors are frequently associated with neurocognitive impairment, cerebral palsy (CP), and seizure disorders. The events leading to perinatal brain injury are multifactorial. This review describes how one subinjurious factor affecting the brain sensitizes it to a second injurious factor, causing an exacerbated injurious cascade. We will review the clinical and experimental evidence, including observations of high rates of maternal and fetal infections in term-born infants with neonatal encephalopathy and cerebral palsy. In addition, we will discuss preclinical evidence for the sensitizing effects of inflammation on injuries, such as hypoxia-ischaemia, our current understanding of the mechanisms underpinning the sensitization process, and the possibility for neuroprotection.

A dual role for AMP-activated protein kinase (AMPK) during neonatal hypoxic-ischaemic brain injury in mice.

Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKß-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling and cell death in the immature central nervous system after hypoxia-ischemia and inflammation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.

Molecular mechanisms of neonatal brain injury.

Fetal/neonatal brain injury is an important cause of neurological disability. Hypoxia-ischemia and excitotoxicity are considered important insults, and, in spite of their acute nature, brain injury develops over a protracted time period during the primary, secondary, and tertiary phases. The concept that most of the injury develops with a delay after the insult makes it possible to provide effective neuroprotective treatment after the insult. Indeed, hypothermia applied within 6 hours after birth in neonatal encephalopathy reduces neurological disability in clinical trials. In order to develop the next generation of treatment, we need to know more about the pathophysiological mechanism during the secondary and tertiary phases of injury. We review some of the critical molecular events related to mitochondrial dysfunction and apoptosis during the secondary phase and report some recent evidence that intervention may be feasible also days-weeks after the insult.

Mitochondrial dysfunction and oxidative and endoplasmic reticulum stress in argyrophilic grain disease.

Argyrophilic grain disease (AGD) is characterized by the accumulation of hyperphosphorylated 4R tau in dendritic varicosities (i.e."grains") in neurons and pretangles in certain areas of the cerebral cortex and other brain regions. We investigated oxidative and endoplasmic reticulum (ER) stress and dysregulation of mitochondrialbiogenesis as potential mechanisms involved in the AGD pathogenesis. Samples from AGD patients (n = 8) and nonpathologic, age-matched controls (n = 5) were compared using biochemical and immunohistochemical techniques with a panel of antibodies to markers of ER stress responses, stress chaperones, oxidative stress and associated cellular responses, respiratory chain complexes, mitochondrial regulators, and modulators of mitochondrial biogenesis. Because AGD is often associated with other tauopathies, mainly Alzheimer disease (AD), results were also compared with those of a group of similar Braak AD stage cases without grains (n = 5). In both AD and AGD cases, we found activation of key molecules that are involved in the unfolded protein response and lead to elevated ER chaperone levels, increased oxidative stress damage, mainly related to lipoxidation and targeting glycolytic enzymes. Altered expression of components of the respiratory chain markers modulating mitochondrial biogenesis were selectively affected in AGD. The findings suggest that, despite the common pathogenic trends in AD and AGD, there is molecular specificity for AGD.

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease.

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.

Cell death and learning impairment in mice caused by in vitro modified pro-NGF can be related to its increased oxidative modifications in Alzheimer disease.

Pro-nerve growth factor (pro-NGF) is expressed at increased levels in Alzheimer's disease (AD)-affected brains and is able to induce cell death in cultures; however, the reasons for these phenomena remain elusive. Here we show that pro-NGF in human AD-affected hippocampus and entorhinal cortex is modified by advanced glycation and lipoxidation end-products in a stage-dependent manner. These modifications block pro-NGF processing to mature NGF, thus making the proneurotrophin especially effective in inducing apoptosis of PC12 cells in culture through the p75 neurotrophin receptor. The processing of advanced glycation and lipoxidation end-products in vitro modified recombinant human pro-NGF is severely impaired, as evidenced by Western blot and by examining its physiological functionality in cell cultures. We also report that modified recombinant human pro-NGF, as well as pro-NGF isolated from human brain affected by AD, cause impairment of learning tasks when administered intracerebroventricularly in mice, which correlates with AD-associated learning impairment. Taken together, the data we present here offer a novel pathway of ethiopathogenesis in AD caused by advanced glycation and lipoxidation end-products modification of pro-NGF.

Pro-NGF from Alzheimer's disease and normal human brain displays distinctive abilities to induce processing and nuclear translocation of intracellular domain of p75NTR and apoptosis.

The pro form of neurotrophic growth factor (pro-NGF), purified by chromatography from human Alzheimer's disease (AD)-affected brains (ADhbi-pro-NGF), has been shown to induce apoptotic cell death in neuronal cell cultures through its interaction with the p75 neurotrophin receptor (p75NTR). In the present work, we report that ADhbi-pro-NGF stimulates processing of p75NTR with alpha- and gamma-secretases, yielding a 20-kd intracellular domain (p75(ICD)) that translocates to the nucleus. This process was accompanied by delayed apoptosis. In AD, p75(ICD) was significantly increased in human entorhinal cortex. Although human frontal cortex has been described as showing a higher pro-NGF increase in AD, the increase in the entorhinal cortex paralleled p75NTR processing in its intracellular domain. In addition, pro-NGF isolated from AD-affected brains differed functionally from pro-NGF isolated from comparably aged control brains, with pro-NGF isolated from control brains being unstable and undergoing degradation to NGF when added to cell culture. As p75(ICD) and pro-NGF are both mediators of apoptosis and are both found in increased levels in the cerebral cortex in AD, the present data have implications for understanding neuronal degeneration in AD.