Nicotine modulates neurogenesis in the central canal during experimental autoimmune encephalomyelitis.

Nicotine has been shown to attenuate experimental autoimmune encephalomyelitis (EAE) through inhibiting inflammation in microglial populations during the disease course. In this study, we investigated whether nicotine modified the regenerative process in EAE by examining nestin-expressing neural stem cells (NSCs) in the spinal cord, which is the primary area of demyelination and inflammation in EAE. Our results show that the endogenous neurogenic responses in the spinal cord after EAE are limited and delayed: while nestin expression is increased, the proliferation of ependymal cells is inhibited compared to healthy animals. Nicotine application significantly reduced nestin expression and partially allowed for the proliferation of ependymal cells. We found that reduction of ependymal cell proliferation correlated with inflammation in the same area, which was relieved by the administration of nicotine. Further, increased numbers of oligodendrocytes (OLs) were observed after nicotine treatment. These findings give a new insight into the mechanism of how nicotine functions to attenuate EAE.

Neurotrophin-3 modulates breast cancer cells and the microenvironment to promote the growth of breast cancer brain metastasis.

Metastasis, which remains incompletely characterized at the molecular and biochemical levels, is a highly specific process. Despite the ability of disseminated cancer cells to intravasate into distant tissues, it has been long recognized that only a limited subset of target organs develop clinically overt metastases. Therefore, subsequent adaptation of disseminated cancer cells to foreign tissue microenvironment determines the metastatic latency and tissue tropism of these cells. As a result, studying interactions between the disseminated cancer cells and the adjacent stromal cells will provide a better understanding of what constitutes a favorable or unfavorable microenvironment for disseminated cancer cells in a tissue-specific manner. Previously, we reported a protein signature of brain metastasis showing increased ability of brain metastatic breast cancer cells to counteract oxidative stress. In this study, we showed that another protein from the brain metastatic protein signature, neurotrophin-3 (NT-3), has a dual function of regulating the metastatic growth of metastatic breast cancer cells and reducing the activation of immune response in the brain. More importantly, increased NT-3 secretion in metastatic breast cancer cells results in a reversion of mesenchymal-like (EMT) state to epithelial-like (MET) state and vice versa. Ectopic expression of NT-3 in EMT-like breast cancer cells reduces their migratory ability and increases the expression of HER2 (human epidermal growth factor receptor 2) and E-cadherin at the cell-cell junction. In addition, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In summary, NT-3 appears to promote growth of metastatic breast cancer cells in the brain by facilitating the re-epithelialization of metastatic breast cancer cells and downmodulating the cytotoxic response of microglia. Most importantly, our results provide new insights into the latency and development of central nervous system macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis.

p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis.

The p53 family member p73 is essential for brain development, but its precise role and scope remain unclear. Global p73 deficiency determines an overt and highly penetrant brain phenotype marked by cortical hypoplasia with ensuing hydrocephalus and hippocampal dysgenesis. The ΔNp73 isoform is known to function as a prosurvival factor of mature postmitotic neurons. In this study, we define a novel essential role of p73 in the regulation of the neural stem cell compartment. In both embryonic and adult neurogenesis, p73 has a critical role in maintaining an adequate neurogenic pool by promoting self-renewal and proliferation and inhibiting premature senescence of neural stem and early progenitor cells. Thus, products of the p73 gene locus are essential maintenance factors in the central nervous system, whose broad action stretches across the entire differentiation arch from stem cells to mature postmitotic neurons.

Microglial activation and intracerebral hemorrhage.

INTRODUCTION: Microglia activate upon injury, migrate to the injury site, proliferate locally, undergo morphological and gene expression changes, and phagocytose injured and dying cells. Cytokines and proteases secreted by these cells contribute to the injury and edema formed. We studied the injury outcome after local elimination/paralysis of microglia. METHODS: Adult male mice were subjected to intracerebral hemorrhage (ICH) by intra-caudate injection of either collagenase or autologous blood. Mice survived for different periods of time, and were subsequently evaluated for neurological deficits, size of the hematoma, and microglia activation. Mice expressing an fms-GFP transgene or the CD11b-HSVTK transgene were also used. For elimination of monocytes/macrophages, CD11b-HSVTK mice were treated with ganciclovir prior to hemorrhage. Modifiers of microglial activation were also used. RESULTS: Induction of ICH resulted in robust microglia activation and recruitment of macrophages. Inactivation of these cells, genetically or pharmacologically, pointed to a critical role of the time of such inactivation, indicating that their role is distinct at different time points following injury. Edema formation is decreased when microglia activation is inhibited, and neurological outcomes are improved. CONCLUSIONS: Microglia, as immunomodulatory cells, have the ability to modify the final presentation of ICH.

Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies.

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.

Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.

Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death.

Reduced cortical injury and edema in tissue plasminogen activator knockout mice after brain trauma.

Tissue plasminogen activator (tPA) may play a deleterious role after brain injury. Here, we compared the response to traumatic brain injury in tPA knockout (KO) and wildtype (WT) mice after controlled cortical impact. At 6 h after trauma, blood-brain barrier permeability was equally increased in all mice. However, by 24 h specific gravity measurements of brain edema were significantly worse in WT mice than in KO mice. At 1 and 2 days post-trauma, mice showed deficits in rotarod performance, but by day 7 all mice recovered motor function and there were no differences between WT and KO mice. At 7 days, cortical lesion volumes were significantly reduced in KO mice compared with WT mice. However, there were no significant differences in CA3 hippocampal neuron survival. These data suggest that tPA amplifies cortical brain damage and edema in this mouse model of traumatic brain injury.

The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system.

Tissue plasminogen activator mediates excitotoxin-induced neurodegeneration and microglial activation in the mouse hippocampus. Here we show that tissue plasminogen activator (tPA) acts in a protease-independent manner to modulate the activation of microglia, the cells of the central nervous system with macrophage properties. Cultured microglia from tPA-deficient mice can phagocytose as efficiently as wild-type microglia. However, tPA-deficient microglia in mixed cortical cultures exhibit attenuated activation in response to lipopolysaccharide, as judged by morphological changes, increased expression of the activation marker F4/80 and the release of the pro-inflammatory cytokine tumor necrosis factor-(&agr;). When tPA is added to tPA deficient cortical cultures prior to endotoxin stimulation, microglial activation is restored to levels comparable to that observed in wild-type cells. Proteolytically-inactive tPA can also restore activation of tPA-deficient microglia in culture and in vivo. However, this inactive enzyme does not restore susceptibility of tPA-deficient hippocampal neurons to excitotoxin-mediated cell death. These results dissociate two different functions of tPA: inactive enzyme can mediate microglial activation, whereas proteolytically-competent protein also promotes neuronal degeneration. Thus tPA is identified as a new cytokine in the central nervous system.

Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus.

BACKGROUND: Injury to the brain induces dramatic local changes in gene expression, cellular morphology and behavior. Activation of microglial cells occurs as an early event after central nervous system (CNS) injury, but it has not been determined whether such activation plays a causal role in neuronal death. We have investigated this question using an excitotoxin-mediated brain injury model system, in conjunction with an endogenous peptide factor (macrophage/microglial inhibiting factor, MIF) that ablates microglial contribution to the cascade. RESULTS: Using MIF, we inhibited the microglial activation that normally follows excitotoxic injury. In cell culture studies, we found that such inhibition blocked the rapid release of microglia-derived tissue plasminogen activator (tPA), an extracellular serine protease made by both neurons and microglia, which we had previously identified as mediating a critical step in excitotoxin-induced neuronal death. Finally, infusion of MIF into the mouse brain prior to excitotoxic insult resulted in the protection of neurons from cell death. CONCLUSIONS: Our results demonstrate that microglia undertake a neurotoxic role when excitotoxic injury occurs in the CNS. They also suggest that the tPA released from microglia has a critical role in triggering neurodegeneration.

Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.

Intravenous tissue plasminogen activator (tPA) is used to treat acute stroke because of its thrombolytic activity and its ability to restore circulation to the brain. However, this protease also promotes neurodegeneration after intracerebral injection of excitotoxins such as glutamate, and neuronal damage after a cerebral infarct is thought to be mediated by excitotoxins. To investigate the effects of tPA on cerebral viability during ischemia/reperfusion, we occluded the middle cerebral artery in wild-type and tPA-deficient mice with an intravascular filament. This procedure allowed us to examine the role of tPA in ischemia, independent of its effect as a thrombolytic agent. tPA-deficient mice exhibited approximately 50% smaller cerebral infarcts than wild-type mice. Intravenous injection of tPA into tPA-/- or wild-type mice produced larger infarcts, indicating that tPA can increase stroke-induced injury. Since tPA promotes desirable (thrombolytic) as well as undesirable (neurotoxic) outcomes during stroke, future therapies should be aimed at countering the excitotoxic damage of tPA to afford even better neuroprotection after an acute cerebral infarct.

Removal of tissue plasminogen activator does not protect against neuronal degeneration in the cerebellum of the weaver mouse.

Tissue plasminogen activator (tPA) is a serine protease that has been shown to be involved in neuronal degeneration. Recently, elevated cerebellar tPA has been reported in a naturally occurring mutant mouse, weaver. Weaver mice suffer extensive degeneration of cerebellar granular neurons during development, leading to severe malformation of the cerebellum as well as abnormal behavior (ataxia). The observations that the developing weaver cerebellum displays a 10-fold increase in tPA activity over wild-type and that a serine protease inhibitor was able to rescue weaver granule cells from premature death in culture suggested that tPA might mediate the death of these mutant neurons. We tested this possibility by introducing the weaver mutation into tPA-deficient mice and comparing the weaver phenotype in the presence or absence of tPA. Analysis at 28 days after birth indicates that tPA-deficient weaver mice are indistinguishable from tPA-containing weaver mice in behavior, cerebellar anatomy, histology, and laminin expression (also reported to be increased in weaver). These results suggest that removal of tPA activity from weaver mice does not protect against neuronal degeneration in the cerebellum and, thus, tPA does not appear to mediate this form of cell death.

Cloning and expression analysis of murine phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells.

Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin.

Mice deficient for plasminogen exhibit a variety of pathologies, all of which examined to date are reversed when the animals are also made fibrin(ogen) deficient. These results suggested that the predominant, and perhaps exclusive, physiological role of plasminogen is clearance of fibrin. Plasminogen-deficient mice also display resistance to excitotoxin-induced neurodegeneration, in contrast with wild-type mice, which are sensitive. Based on the genetic interaction between plasminogen and fibrinogen, we investigated whether resistance to neuronal cell death in the plasminogen-deficient mice is dependent on fibrin(ogen). Unexpectedly, mice lacking both plasminogen and fibrinogen are resistant to neurodegeneration to levels comparable to plasminogen-deficient mice. Therefore, plasmin acts on substrates other than fibrin during experimental neuronal degeneration, and may function similarly in other pathological settings in the central nervous system.

Clinical implications of the involvement of tPA in neuronal cell death.

Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.

An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus.

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.

Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator.

Tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to active plasmin, is produced in the rat and mouse hippocampus and participates in neuronal plasticity. To help define the role of tPA in the nervous system, we have analyzed the regulation of its expression in the neuronal cell line PC12. In control cultures, tPA activity is exclusively cell-associated, and no activity is measurable in the culture medium. When the cells are treated with depolarizing agents, such as KCI, tPA activity becomes detectable in the medium. The increased secreted tPA activity is not accompanied by an increase in tPA mRNA levels, and it is not blocked by protein synthesis inhibitors. In contrast, tPA release is abolished by Ca2+ channel blockers, suggesting that chemically induced membrane depolarization stimulates the secretion of preformed enzyme. Moreover, KCI has a similar effect in vivo when administered to the murine brain via an osmotic pump: tPA activity increases along the CA2-CA3 regions and dentate gyrus of the hippocampal formation. These results demonstrate a neuronal activity-dependent secretory mechanism that can rapidly increase the amount of tPA in neuronal tissue.

Isolation and characterization of two novel, cytoplasmically polyadenylated, oocyte-specific, mouse maternal RNAs.

During early development in mouse and Xenopus, translational activation of stored maternal mRNAs by cytoplasmic polyadenylation requires both the nuclear polyadenylation signal AAUAAA and U-rich cis-acting adenylation control elements (ACEs), also termed cytoplasmic polyadenylation elements, located in the 3' UTR. Using an ACE-based PCR strategy (Sallés et al., 1992) we have isolated two novel cDNAs from mouse oocytes: OM2a and OM2b (for Oocyte Maturation). Each message contains an ACE consensus sequence upstream of AAUAAA, is specifically transcribed in the growing oocyte, and is cytoplasmically polyadenylated upon oocyte maturation. Comparison of the mouse and rat homologs reveals considerable nucleotide sequence homology and conservation of overall gene organization. However, the predicted open reading frames are far less conserved, suggesting that these genes may not be functioning as proteins. The tissue specificity and tight temporal regulation of the RNAs suggest a role for these genes during early development.