CCR2+ monocytes replenish border-associated macrophages in the diseased mouse brain.

Border-associated macrophages (BAMs) are tissue-resident macrophages that reside at the border of the central nervous system (CNS). Since BAMs originate from yolk sac progenitors that do not persist after birth, the means by which this population of cells is maintained is not well understood. Using two-photon microscopy and multiple lineage-tracing strategies, we determine that CCR2+ monocytes are significant contributors to BAM populations following disruptions of CNS homeostasis in adult mice. After BAM depletion, while the residual BAMs possess partial self-repopulation capability, the CCR2+ monocytes are a critical source of the repopulated BAMs. In addition, we demonstrate the existence of CCR2+ monocyte-derived long-lived BAMs in a brain compression model and in a sepsis model after the initial disruption of homeostasis. Our study reveals that the short-lived CCR2+ monocytes transform into long-lived BAM-like cells at the CNS border and subsequently contribute to BAM populations.

Integration of single-cell transcriptomes and biological function reveals distinct behavioral patterns in bone marrow endothelium.

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue's homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs' ECs to decode mechanistic information and explore therapeutics.

Fate mapping via CCR2-CreER mice reveals monocyte-to-microglia transition in development and neonatal stroke.

Whether monocytes contribute to the brain microglial pool in development or after brain injury remains contentious. To address this issue, we generated CCR2-CreER mice to track monocyte derivatives in a tamoxifen-inducible manner. This method labeled Ly6Chi and Ly6Clo monocytes after tamoxifen dosing and detected a surge of perivascular macrophages before blood-brain barrier breakdown in adult stroke. When dosed by tamoxifen at embryonic day 17 (E17), this method captured fetal hematopoietic cells at E18, subdural Ki67+ ameboid cells at postnatal day 2 (P2), and perivascular microglia, leptomeningeal macrophages, and Iba1+Tmem119+P2RY12+ parenchymal microglia in selective brain regions at P24. Furthermore, this fate mapping strategy revealed an acute influx of monocytes after neonatal stroke, which gradually transformed into a ramified morphology and expressed microglial marker genes (Sall1, Tmem119, and P2RY12) for at least 62 days after injury. These results suggest an underappreciated level of monocyte-to-microglia transition in development and after neonatal stroke.

KRasG12D expression in the bone marrow vascular niche affects hematopoiesis with inflammatory signals.

The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have indicated that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping polymerase chain reaction revealed KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly fewer functional hematopoietic stem cells, and immunofluorescence imaging showed that the hematopoietic stem cells in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA sequencing analyses found an inflammatory gene network, especially tumor necrosis factor α, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.

Microglial-mediated PDGF-CC activation increases cerebrovascular permeability during ischemic stroke.

Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1-/-) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1-/- mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b+ cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1-/- mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.

Osteopontin Is a Blood Biomarker for Microglial Activation and Brain Injury in Experimental Hypoxic-Ischemic Encephalopathy.

Clinical management of neonatal hypoxic-ischemic encephalopathy (HIE) suffers from the lack of reliable surrogate marker tests. Proteomic analysis may identify such biomarkers in blood, but there has been no proof-of-principle evidence to support this approach. Here we performed in-gel trypsin digestion of plasma proteins from four groups of 10-d-old mice [untouched and 24 h after low-dose lipopolysaccharide (LPS) exposure, hypoxia-ischemia (HI), or LPS/HI injury; n = 3 in each group) followed by liquid chromatography-tandem mass spectrometry and bioinformatics analysis to search for HI- and LPS/HI-associated brain injury biomarkers. This analysis suggested the induction of plasma osteopontin (OPN) by HI and LPS/HI, but not by sham and injury-free LPS exposure. Immunoblot confirmed post-HI induction of OPN protein in brain and blood, whereas Opn mRNA was induced in brain but not in blood. This disparity suggests brain-derived plasma OPN after HI injury. Similarly, immunostaining showed the expression of OPN by Iba1+ microglia/macrophages in HI-injured brains. Further, intracerebroventricular injection of LPS activated microglia and up-regulated plasma OPN protein. Importantly, the induction of plasma OPN after HI was greater than that of matrix metalloproteinase 9 or glial fibrillary acid protein. Plasma OPN levels at 48 h post-HI also parallel the severity of brain damage at 7-d recovery. Together, these results suggest that OPN may be a prognostic blood biomarker in HIE through monitoring brain microglial activation.

Aryl hydrocarbon receptor mediates both proinflammatory and anti-inflammatory effects in lipopolysaccharide-activated microglia.

The aryl hydrocarbon receptor (AhR) regulates peripheral immunity; but its role in microglia-mediated neuroinflammation in the brain remains unknown. Here, we demonstrate that AhR mediates both anti-inflammatory and proinflammatory effects in lipopolysaccharide (LPS)-activated microglia. Activation of AhR by its ligands, formylindolo[3,2-b]carbazole (FICZ) or 3-methylcholanthrene (3MC), attenuated LPS-induced microglial immune responses. AhR also showed proinflammatory effects, as evidenced by the findings that genetic silence of AhR ameliorated the LPS-induced microglial immune responses and LPS-activated microglia-mediated neurotoxicity. Similarly, LPS-induced expressions of tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were reduced in the cerebral cortex of AhR-deficient mice. Intriguingly, LPS upregulated and activated AhR in the absence of AhR ligands via the MEK1/2 signaling pathway, which effects were associated with a transient inhibition of cytochrome P450 1A1 (CYP1A1). Although AhR ligands synergistically enhance LPS-induced AhR activation, leading to suppression of LPS-induced microglial immune responses, they cannot do so on their own in microglia. Chromatin immunoprecipitation results further revealed that LPS-FICZ co-treatment, but not LPS alone, not only resulted in co-recruitment of both AhR and NFκB onto the κB site of TNFα gene promoter but also reduced LPS-induced AhR binding to the DRE site of iNOS gene promoter. Together, we provide evidence showing that microglial AhR, which can be activated by LPS, exerts bi-directional effects on the regulation of LPS-induced neuroinflammation, depending on the availability of external AhR ligands. These findings confer further insights into the potential link between environmental factors and the inflammatory brain disorders.

Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α) through inhibition of prolyl hydrolase 2 (PHD2) and activation of the phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo) and vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

Overexpression of vascular endothelial growth factor in the germinal matrix induces neurovascular proteases and intraventricular hemorrhage.

Intracranial hemorrhage in preterm neonates may result in neonatal mortality and functional disabilities, but its pathogenic mechanisms are poorly defined and better therapies are needed. We used a tetracycline-regulated transgenic system to test whether the induction of vascular endothelial growth factor (VEGF) in the germinal matrix leads to intracranial hemorrhage. This genetic strategy initially induced a dense network of loosely adjoined endothelial cells and pericytes near lateral ventricles, similar to the immature vascular rete in human fetal brains. Yet, this rich vascular network transformed into low-vasculature patches correlated with hemorrhage and caspase-3 activation near birth. Gene expression and biochemical analyses suggested that downstream mediators of VEGF in this network include transcriptional factors ETS1 and HIF2α (hypoxia-inducible factor 2α), components of the PDGFß (platelet-derived growth factor ß) and TGFß (transforming growth factor-ß) receptor signaling pathways, matrix metalloproteinase-9 (MMP-9), and cathepsins. Prenatal administration of glucocorticoids markedly reduced mortality and cerebral hemorrhage in mutant animals, as in human neonates. This protective effect was not due to blocking vasculogenesis, but was instead associated with inhibition of neurovascular proteases, notably MMP-9, cathepsin B, and caspase-3. Collectively, these results support a causative role of VEGF in perinatal cerebral hemorrhage and implicate its downstream proteases as potential therapeutic targets.

Intranasal delivery of cell-penetrating anti-NF-κB peptides (Tat-NBD) alleviates infection-sensitized hypoxic-ischemic brain injury.

Perinatal infection aggravates neonatal hypoxic-ischemic (HI) brain injury and may interfere with therapeutic hypothermia. While the NF-κB signaling pathway has been implicated in microglia activation in infection-sensitized HI, the current therapeutic strategies rely on systemic intervention, which could impair neonatal immunity and increase the risk of severe infection. To devise a brain-targeted anti-NF-κB strategy, we examined the effects of intranasal delivery of tat-NBD peptides in two animal models of neonatal infection-sensitized HI. Kinetic experiments showed that tat-NBD peptides entered the olfactory bulbs rapidly (10-30 min) and peaked in the cerebral cortex around 60 min after intranasal application in P7 rats. Further, intranasal delivery of 1.4 mg/kg tat-NBD, which is only 7% of the intravenous dose in past studies, markedly attenuated NF-κB signaling, microglia activation, and brain damage triggered by HI with 4 or 72 h pre-exposure to the bacterial endotoxin lipopolysaccharide (LPS). In contrast, intranasal delivery of mutant tat-NBD peptides or systemic application of minocycline failed to block LPS-sensitized HI injury. Yet, intranasal delivery of up to 5.6 mg/kg tat-NBD peptides immediately after pure-HI insult showed little protection, likely due to its rapid clearance from the brain and inability to inhibit parenchymal plasminogen activators. Together, these results suggest a novel therapy of infection-sensitized HI brain injury in newborns.

Loss of RhoA in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation.

The organization of neural progenitors in the developing mammalian neuroepithelium is marked by cadherin-based adherens junctions. Whereas RhoA, a founding member of the small Rho GTPase family, has been shown to play important roles in epithelial adherens junctions, its physiological roles in neural development remain uncertain due to the lack of specific loss-of-function studies. Here, we show that RhoA protein accumulates at adherens junctions in the developing mouse brain and colocalizes to the cadherin-catenin complex. Conditional deletion of RhoA in midbrain and forebrain neural progenitors using Wnt1-Cre and Foxg1-Cre mice, respectively, disrupts apical adherens junctions and causes massive dysplasia of the brain. Furthermore, RhoA-deficient neural progenitor cells exhibit accelerated proliferation, reduction of cell- cycle exit, and increased expression of downstream target genes of the hedgehog pathway. Consequently, both lines of conditional RhoA-deficient embryos exhibit expansion of neural progenitor cells and exencephaly-like protrusions. These results demonstrate a critical role of RhoA in the maintenance of apical adherens junctions and the regulation of neural progenitor proliferation in the developing mammalian brain.

Developmental and post-injury cortical gliogenesis: a genetic fate-mapping study with Nestin-CreER mice.

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin-CreER mice to fate-map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin-CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop-floxed EGFP mice and receive tamoxifen in late embryogenesis (E16-E18). Quantification showed E18 tamoxifen-induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time-lapse microscopy showed SVZ-glioblasts underwent complex metamorphosis and often-reciprocal transformation into transitional RG. Surprisingly, the E10-dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18-induction fate-mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate-mapping and BrdU-labeling, we showed that cortical astrocytes cease proliferation soon after birth (

Coordinated control of self-renewal and differentiation of neural stem cells by Myc and the p19ARF-p53 pathway.

The modes of proliferation and differentiation of neural stem cells (NSCs) are coordinately controlled during development, but the underlying mechanisms remain largely unknown. In this study, we show that the protooncoprotein Myc and the tumor suppressor p19(ARF) regulate both NSC self-renewal and their neuronal and glial fate in a developmental stage-dependent manner. Early-stage NSCs have low p19(ARF) expression and retain a high self-renewal and neurogenic capacity, whereas late-stage NSCs with higher p19(ARF) expression possess a lower self-renewal capacity and predominantly generate glia. Overexpression of Myc or inactivation of p19(ARF) reverts the properties of late-stage NSCs to those of early-stage cells. Conversely, inactivation of Myc or forced p19(ARF) expression attenuates self-renewal and induces precocious gliogenesis through modulation of the responsiveness to gliogenic signals. These actions of p19(ARF) in NSCs are mainly mediated by p53. We propose that opposing actions of Myc and the p19(ARF)-p53 pathway have important functions in coordinated developmental control of self-renewal and cell fate choices in NSCs.

Deleterious effects of plasminogen activators in neonatal cerebral hypoxia-ischemia.

The immature brains of newborns often respond differently from the brains of adults when exposed to similar insults. Previous studies have indicated that although hypoxia-ischemia (HI) induces persistent thrombosis in adult brains, it only modestly impairs blood perfusion in newborn brains. Here, we used the Vannucci model of HI encephalopathy to study age-related responses to cerebral HI in rat pups. We found that HI triggered fibrin deposition and impaired blood perfusion in both neonatal and adult brains. However, these effects were only transient in neonatal brains (<4 hours) and were accompanied by acute induction of both tissue-type and urinary-type plasminogen activators (tPA and uPA), which was not observed in adult brains subjected to the same insult. Interestingly, activation of the plasminogen system persisted up to 24 hours in neonatal brains, long after the clearance of fibrin-rich thrombi. Furthermore, astrocytes and macrophages outside blood vessels expressed tPA after HI, suggesting the possibility of tPA/plasmin-mediated cytotoxicity. Consistent with this hypothesis, injection of alpha2-antiplasmin into cerebral ventricles markedly ameliorated HI-induced damage to neurofilaments and white matter oligodendrocytes, providing a dose-response reduction of brain injury after 7 days of recovery. Conversely, ventricular injection of tPA increased HI-induced brain damage. Together, these results suggest that tPA/plasmin induction, which may contribute to acute fibrinolysis, is a critical component of extravascular proteolytic damage in immature brains, representing a new therapeutic target for the treatment of HI encephalopathy.

Nestin-CreER mice reveal DNA synthesis by nonapoptotic neurons following cerebral ischemia hypoxia.

The standard method of detecting neurogenesis uses bromodeoxyuridine (BrdU) to label DNA synthesis followed by double labeling with neuronal markers. However, DNA synthesis may occur in events unrelated to neurogenesis including aneuploidy and abortive cell cycle reentry. Hence, it is important to confirm neurogenesis with methods other than BrdU incorporation. To this end, we have generated transgenic nestin-CreER mice that express tamoxifen-inducible Cre recombinase under the control of a nestin enhancer. When crossed with a ubiquitous Enhanced Green Fluorescent Protein (EGFP)-Cre-reporter line, the bitransgenic animals can reveal the nestin-positive progenitors and their progeny with EGFP after tamoxifen induction. This system has many applications including visualization of embryonic neural progenitors, detection of postnatally transformed radial glial cells, and labeling adult neural progenitors in the subventricular zone (SVZ). To examine the contribution of SVZ progenitors to cell replacement after stroke, tamoxifen-induced mice were challenged with focal ischemia or combined ischemia-hypoxia followed by BrdU injection. This analysis revealed only very few EGFP-positive cells outside the SVZ after focal ischemia but robust DNA synthesis by hippocampal neurons without immediate cell death following ischemia-hypoxia. These results suggest that the nestin-CreER system is a useful tool for detecting embryonic and adult neurogensis. They also confirm the existence of nonproliferative DNA synthesis by old neurons after experimental brain injury.

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells.

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.

Mixed lineage kinase-c-jun N-terminal kinase signaling pathway: a new therapeutic target in Parkinson's disease.

There is growing evidence that the molecular pathways of programmed cell death play a role in neurodegenerative disease, including Parkinson's disease, so there has been increased interest in them as therapeutic targets for the development of neuroprotective strategies. One pathway of cell death that has attracted particular attention is the mixed lineage kinase (MLK) -c-jun N-terminal kinase (JNK) signaling cascade, which leads to the phosphorylation and activation of the transcription factor c-jun. There is much evidence, from in vitro and in vivo studies, that this cascade can mediate cell death. In addition, there is evidence that it is operative upstream in the death process. It is possible that abrogation of this pathway may forestall death before irreversible cellular injury. One class of compounds that has shown promise for their ability to block cell death by inhibiting this cascade are the inhibitors of the MLKs, which are upstream in the activation of c-jun. One of these compounds, CEP1347, is now in a Phase II/III clinical trial for neuroprotection in PD. Whether this trial is successful or not, this signaling cascade is likely to be a focus of future therapeutic development. This review, therefore, outlines the principles of signaling within this kinase pathway, and the evidence for its role in cell death. We review the evidence that inhibition of the MLKs can prevent dopamine neuron cell death and the degeneration of their axons. These studies suggest important future directions for the development of therapies that will target this important cell death pathway.

Specific pathophysiological functions of JNK isoforms in the brain.

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c-JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6-hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment-specific expression of JNK isoforms with 46-kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6-hydroxydopamine was transiently improved in JNK3 ko and c-JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c-JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid-induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c-Jun irrespective on the experimental set-up. This comprehensive study provides novel insights into the context-dependent physiological and pathological functions of JNK isoforms.

Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain.

Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia-ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia-ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+ neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia-ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia-ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.