Endocannabinoid Receptor 2 Function is Associated with Tumor-Associated Macrophage Accumulation and Increases in T Cell Number to Initiate a Potent Antitumor Response in a Syngeneic Murine Model of Glioblastoma.

Introduction: Glioblastoma patients have a highly immunosuppressive tumor microenvironment and systemic immunosuppression that comprise a major barrier to immune checkpoint therapy. Based on the production of endocannabinoids by glioblastomas, we explored involvement of endocannabinoid receptor 2 (CB2R), encoded by the CNR2 gene, which is predominantly expressed by immune cells, in glioblastoma-related immunosuppression. Materials & Methods: Bioinformatics of human glioblastoma databases was used to correlate enzymes involved in the synthesis and degradation of endocannabinoids, as well as CB2Rs, with patient overall survival. Intrastriatal administration of luciferase-expressing, murine GL261 glioblastoma cells was used to establish in in vivo glioblastoma model for characterization of tumor growth and intratumoral immune cell infiltration, as well as provide immune cells for in vitro co-culture experiments. Involvement of CB2Rs was determined by treatment with CB2R agonist (GW405833) or CB2R antagonist (AM630). ELISA, FACS, and immunocytochemistry were used to determine perforin, granzyme B, and surface marker levels. Results: Bioinformatics of human glioblastoma databases showed high expression of CB2R and elevated endocannabinoid production correlated with poorer prognosis, and involved immune-associated pathways. AM630treatment of GL261 glioblastoma-bearing mice induced a potent antitumor response, with survival plateauing at 50% on Day 40, when all control mice (median survival 28 days) and mice treated with GW405833 (median survival 21 days) had died. Luciferase tumor imaging revealed accelerated tumor growth by GW405833 treatment, but stable or regressing tumors in AM630-treated mice. Notably, in spleens, AM630 treatment caused an 83% decrease in monocytes/macrophages, and 1.8- and 1.6-fold increases in CD8+ and CD4+ cells, respectively. Within tumors, there was a corresponding decrease in tumor-associated macrophages (TAMs) and increase in CD8+ T cells. In vitro, lymphocytes from AM630-treated mice showed greater cytotoxic function (increased percentage of perforin- and granzyme B-positive CD8+ T cells). Discussion: These results suggest that inhibition of CB2R enhances both immunosuppressive TAM infiltration and systemic T-cell suppression through CB2R activation, and that inhibition of CB2Rs can potently counter both the immunosuppressive tumor microenvironment, as well as systemic immunosuppression in glioblastoma.

Increased SLC7A3 Expression inhibits Tumor Cell Proliferation and Predicts a Favorable Prognosis in Breast Cancer.

BACKGROUND: Arginine plays significant and contrasting roles in breast cancer growth and survival. However, the factors governing arginine balance remain poorly characterized. OBJECTIVE: We aimed to identify the molecule that governs arginine metabolism in breast cancer and to elucidate its significance. METHODS: We analyzed the correlation between the expression of solute carrier family 7 member 3 (SLC7A3), the major arginine transporter, and breast cancer survival in various databases, including GEPIA, UALCAN, Metascape, String, Oncomine, KM-plotter, CBioPortal and Prognosis. Additionally, we validated our findings through bioinformatic analyses and experimental investigations, including colony formation, wound healing, transwell, and mammosphere formation assays. RESULTS: Our analysis revealed a significant reduction in SLC7A3 expression in all breast cancer subtypes compared to adjacent breast tissues. Kaplan-Meier survival analyses demonstrated that high SLC7A3 expression was positively associated with decreased nodal metastasis (HR=0.70, 95% CI [0.55, 0.89]), ER positivity (HR=0.79, 95% CI [0.65, 0.95]), and HER2 negativity (HR=0.69, 95% CI [0.58, 0.82]), and increased recurrence-free survival. Moreover, low SLC7A3 expression predicted poor prognosis in breast cancer patients for overall survival. Additionally, the knockdown of SLC7A3 in MCF-7 and MDA-MB-231 cells resulted in increased cell proliferation and invasion in vitro. CONCLUSION: Our findings indicate a downregulation of SLC7A3 expression in breast cancer tissues compared to adjacent breast tissues. High SLC7A3 expression could serve as a prognostic indicator for favorable outcomes in breast cancer patients due to its inhibitory effects on breast cancer cell proliferation and invasion.

The multifunctional human ocular melanocortin system.

Immune privilege in the eye involves physical barriers, immune regulation and secreted proteins that together limit the damaging effects of intraocular immune responses and inflammation. The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) normally circulates in the aqueous humour of the anterior chamber and the vitreous fluid, secreted by iris and ciliary epithelium, and retinal pigment epithelium (RPE). α-MSH plays an important role in maintaining ocular immune privilege by helping the development of suppressor immune cells and by activating regulatory T-cells. α-MSH functions by binding to and activating melanocortin receptors (MC1R to MC5R) and receptor accessory proteins (MRAPs) that work in concert with antagonists, otherwise known as the melanocortin system. As well as controlling immune responses and inflammation, a broad range of biological functions is increasingly recognised to be orchestrated by the melanocortin system within ocular tissues. This includes maintaining corneal transparency and immune privilege by limiting corneal (lymph)angiogenesis, sustaining corneal epithelial integrity, protecting corneal endothelium and potentially enhancing corneal graft survival, regulating aqueous tear secretion with implications for dry eye disease, facilitating retinal homeostasis via maintaining blood-retinal barriers, providing neuroprotection in the retina, and controlling abnormal new vessel growth in the choroid and retina. The role of melanocortin signalling in uveal melanocyte melanogenesis however remains unclear compared to its established role in skin melanogenesis. The early application of a melanocortin agonist to downregulate systemic inflammation used adrenocorticotropic hormone (ACTH)-based repository cortisone injection (RCI), but adverse side effects including hypertension, edema, and weight gain, related to increased adrenal gland corticosteroid production, impacted clinical uptake. Compared to ACTH, melanocortin peptides that target MC1R, MC3R, MC4R and/or MC5R, but not adrenal gland MC2R, induce minimal corticosteroid production with fewer adverse systemic effects. Pharmacological advances in synthesising MCR-specific targeted peptides provide further opportunities for treating ocular (and systemic) inflammatory diseases. Following from these observations and a renewed clinical and pharmacological interest in the diverse biological roles of the melanocortin system, this review highlights the physiological and disease-related involvement of this system within human eye tissues. We also review the emerging benefits and versatility of melanocortin receptor targeted peptides as non-steroidal alternatives for inflammatory eye diseases such as non-infectious uveitis and dry eye disease, and translational applications in promoting ocular homeostasis, for example, in corneal transplantation and diabetic retinopathy.

A fragment of cell adhesion molecule L1 reduces amyloid-ß plaques in a mouse model of Alzheimer's disease.

Deposition of amyloid-ß (Aß) in the brain is one of the important histopathological features of Alzheimer's disease (AD). Previously, we reported a correlation between cell adhesion molecule L1 (L1) expression and the occurrence of AD, but its relationship was unclear. Here, we report that the expression of L1 and a 70 kDa cleavage product of L1 (L1-70) was reduced in the hippocampus of AD (APPswe) mice. Interestingly, upregulation of L1-70 expression in the hippocampus of 18-month-old APPswe mice, by parabiosis involving the joining of the circulatory system of an 18-month-old APPswe mouse with a 2-month-old wild-type C57BL/6 mouse, reduced amyloid plaque deposition. Furthermore, the reduction was accompanied by the appearance of a high number of activated microglia. Mechanistically, we observed that L1-70 could combine with topoisomerase 1 (Top1) to form a complex, L1-70/Top1, that was able to regulate expression of macrophage migration inhibitory factor (MIF), resulting in the activation of microglia and reduction of Aß plaques. Also, transforming growth factor ß1 (TGFß-1) transferred from the blood of young wild-type C57BL/6 mice to the aged AD mice, was identified as a circulating factor that induces full-length L1 and L1-70 expression. All together, these findings suggest that L1-70 contributes to the clearance of Aß in AD, thereby adding a novel perspective in understanding AD pathogenesis.

Duloxetine ameliorates valproic acid-induced hyperactivity, anxiety-like behavior, and social interaction deficits in zebrafish.

Syndromic autism spectrum disorders (ASDs) are characterized by impaired social communication and repetitive/stereotyped behaviors. Currently available therapeutic agents against ASD have limited efficacy. Thus, searching for novel and effective drugs ameliorating core symptoms, in particular social deficits, is of utmost importance. Duloxetine (DLX), an antidepressant that has been identified as an agonist mimetic for the cell adhesion molecule L1, exhibits beneficial functions in vitro and in vivo. Therefore, in this study, we focused on the rapid and persistent neuroprotective function of DLX following valproic acid (VPA)-triggered hyperactivity, anxiety-like behavior and social deficits in zebrafish. Embryonic exposure to VPA reduced survival in a dose- and time-dependent manner, delayed hatching, and also resulted in a significant number of malformed larvae. After initial dose-response experiments in zebrafish larvae, 10 µM VPA exposure between 0.33 and 4.5 days post fertilization (dpf) was identified as an effective concentration that led to an early and persistent ASD-like phenotype in zebrafish. ASD-like elevated acetylcholine esterase (AChE) activity and reduced Akt-mTOR signaling was observed in zebrafish whole brain. Acute administration of DLX (4.5-6 dpf) reduced the VPA-induced ASD-like phenotype in zebrafish larvae. Additionally, such early-life acute DLX treatment had long-term effects in ameliorating social impairments, hyperactivity, and anxiety-like behaviors through adulthood. This was accompanied by reduced AChE activity and by normalized Akt-mTOR signaling. Overall, DLX treatment showed a long-term therapeutic effect on autistic-like behaviors, and alteration of AChE activity and Akt-mTOR signaling were identified as crucial in the VPA-induced ASD zebrafish model.

Chondroitin 6-sulfate-binding peptides improve recovery in spinal cord-injured mice.

The role of glycosaminoglycan sulfation patterns, particularly in regard to scar formation and inhibition of neuritogenesis, has been mainly studied in cell culture with a focus on chondroitin 4-sulfate. In this study, we investigated chondroitin 6-sulfate (C6S) and found that it also inhibits neurite outgrowth of mouse cerebellar granule neurons in vitro. To examine whether the inhibitory activity of C6S could be neutralized, seven previously characterized high-affinity C6S-binding peptides were tested, among which three peptides neutralized the inhibitory functions of C6S. We further investigated these peptides in a mouse model of spinal cord injury, since upregulation of C6S expression in the glial scar following injury has been associated with reduced axonal regrowth and functional recovery. We here subjected mice to severe compression injury at thoracic levels T7-T9, immediately followed by inserting gelfoam patches soaked in C6S-binding peptides or in a control peptide. Application of C6S-binding peptides led to functional recovery after injury and prevented fibrotic glial scar formation, as seen by decreased activation of astrocytes and microglia/macrophages. Decreased expression of several lecticans and deposition of fibronectin at the site of injury were also observed. Application of C6S-binding peptides led to axonal regrowth and inhibited the C6S-mediated activation of RhoA/ROCK and decrease of PI3K-Akt-mTOR signaling pathways. Taken together, these results indicate that treatment with C6S-binding peptides improves functional recovery in a mouse model of spinal cord injury.

The role of melanocytes in the human choroidal microenvironment and inflammation: Insights from the transcriptome.

The choroid within the human eye contains a rich milieu of cells including melanocytes. Human choroidal melanocytes (HCMs) absorb light, regulate free radical production, and were recently shown to modulate inflammation. This study aimed to identify key genes and pathways involved in the inflammatory response of HCMs through the use of RNA-seq. Primary HCMs were cultured from donor choroids, RNA was extracted from control and lipopolysaccharide (LPS)-treated HCMs, and mRNA was sequenced. Functional annotation and pathway analysis were performed using gene ontology and gene set enrichment analyses. Representative RNA-seq results were verified with RT-qPCR and protein measurements. We detected 100 differentially expressed genes including an array of CCL and CXCL cytokines and mediators of cell-cell and cell-matrix adhesion, such as ICAM1, CLDN1, CCN3, ITGA1 and ITGA11. Functional annotation showed that these gene sets control inflammatory pathways, immune cell trafficking, cell-cell adhesion, interactions with the extracellular matrix and blood vessels, angiogenesis and epithelial-to-mesenchymal transitions. Our study provides insights into the transcriptional regulation of primary HCMs in response to inflammatory stimuli and identifies novel melanocyte-driven mechanisms potentially involved in choroidal homeostasis and inflammation.

Adhesion Molecule L1 Agonist Mimetics Protect Against the Pesticide Paraquat-Induced Locomotor Deficits and Biochemical Alterations in Zebrafish.

Besides several endogenous elements, exogenous factors, including exposure to pesticides, have been recognized as putative factors contributing to the onset and development of neurodegenerative diseases, including Parkinson's disease (PD). Considering the availability, success rate, and limitations associated with the current arsenals to fight PD, there is an unmet need for novel therapeutic interventions. Therefore, based on the previously reported beneficial functions of the L1 cell adhesion molecule, we hypothesized that L1 mimetic compounds may serve to neutralize neurotoxicity triggered by the pesticide paraquat (PQ). In this study, we attempt to use PQ for inducing PD-like pathology and the L1 mimetic compounds phenelzine sulfate (PS) and tacrine (TC) as potential candidates for the amelioration of PD symptoms using zebrafish as a model system. Administration of PQ together with the L1 mimetic compounds PS or TC (250 nM) improved survival of zebrafish larvae, protected them from locomotor deficits, and increased their sensorimotor reflexes. Moreover, application of PQ together with PS (500 nM) or TC (1000 nM) in adult zebrafish counteracted PQ-induced toxicity, maintaining normal locomotor functions and spatial memory in an open field and T-maze task, respectively. Both L1 mimetic compounds prevented reduction in tyrosine hydroxylase and dopamine levels, reduced reactive oxygen species (ROS) generation, protected against impairment of mitochondrial viability, improved the antioxidant enzyme system, and prevented a decrease in ATP levels. Altogether, our findings highlight the beneficial functions of the agonistic L1 mimetics PS and TC by improving several vital cell functions against PQ-triggered neurotoxicity.

Identification of the distribution of human endogenous retroviruses K (HML-2) by PCR-based target enrichment sequencing.

BACKGROUND: Human endogenous retroviruses (HERVs), suspected to be transposition-defective, may reshape the transcriptional network of the human genome by regulatory elements distributed in their long terminal repeats (LTRs). HERV-K (HML-2), the most preserved group with the least number of accumulated of mutations, has been associated with aberrant gene expression in tumorigenesis and autoimmune diseases. Because of the high sequence similarity between different HERV-Ks, current methods have limitations in providing genome-wide mapping specific for individual HERV-K (HML-2) members, a major barrier in delineating HERV-K (HML-2) function. RESULTS: In an attempt to obtain detailed distribution information of HERV-K (HML-2), we utilized a PCR-based target enrichment sequencing protocol for HERV-K (HML-2) (PTESHK) loci, which not only maps the presence of reference loci, but also identifies non-reference loci, enabling determination of the genome-wide distribution of HERV-K (HML-2) loci. Here we report on the genomic data obtained from three individuals. We identified a total of 978 loci using this method, including 30 new reference loci and 5 non-reference loci. Among the 3 individuals in our study, 14 polymorphic HERV-K (HML-2) loci were identified, and solo-LTR330 and N6p21.32 were identified as polymorphic for the first time. CONCLUSIONS: Interestingly, PTESHK provides an approach for the identification of the genome-wide distribution of HERV-K (HML-2) and can be used for the identification of polymorphic loci. Since polymorphic HERV-K (HML-2) integrations are suspected to be related to various diseases, PTESHK can supplement other emerging techniques in accessing polymorphic HERV-K (HML-2) elements in cancer and autoimmune diseases.

Neuroprotective Effects of Loganin on MPTP-Induced Parkinson's Disease Mice: Neurochemistry, Glial Reaction and Autophagy Studies.

Parkinson's disease (PD) is a progressive neurodegenerative disease, involving resting tremor and bradykinesia, for which no recognized therapies or drugs are available to halt or slow progression. In recent years, natural botanic products have been considered relatively safe, with limited side effects, and are expected to become an important source for clinical mediation of PD in the future. Our study focuses on the ability of loganin, a compound derived from fruits of cornus, to mediate neuroprotection in a mouse model of PD. Mice were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) with a dosage of 30 mg/kg daily for 5 days to establish a subacute PD model and treated with loganin. Locomotor activity was assessed by a pole test, then mice were euthanized at 1 and 3 days after the last treatment, and brain tissue was prepared for subsequent assays. Loganin rescued decrease of dopamine levels and tyrosine hydroxylase (TH) expression in the striatum, and shortened total locomotor activity (TLA) time of mice. Furthermore, loganin alleviated microglia and astrocyte activation, and suppressed TNF-α and caspase-3 expression through a c-Abl-p38-NFκB pathway. Loganin also downregulated LC3-II and Drp1 expression, and decreased the level of acidic vesicular organelles (AVOs). Loganin exerts neuroprotective effects on MPTP-induced PD mice by decreasing inflammation, autophagy, and apoptosis, suggesting that loganin could serve as a therapeutic drug to ameliorate PD. J. Cell. Biochem. 118: 3495-3510, 2017. © 2017 Wiley Periodicals, Inc.

Unexpected Neuroprotective Effects of Loganin on 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Neurotoxicity and Cell Death in Zebrafish.

1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), which induces the pathological characteristics of Parkinson's disease in rodents, also specifically targets dopaminergic neurons in zebrafish embryos and larvae. Loganin, a traditional Chinese drug, was reported to regulate immune function and possess anti-inflammatory and anti-shock effects. Here, we investigate the role of loganin in MPTP-induced Parkinson-like abnormalities in zebrafish. MPTP treatment-induced abnormal development, in larvae, such as pericardium edema, increased yolk color, yolk sac edema, and retarded yolk sac resorption, as well as defects in brain development. Loganin could block MPTP-induced defects, with little toxicity to the eggs. Results of whole mount in situ hybridization showed loganin prevented the loss of both dopaminergic neurons and locomotor activity, exhibited by larvae treated with MPTP. In addition, loganin significantly rescued MPTP-induced neurotoxicity on PC12 cells, possibly through the suppression of PI3K/Akt/mTOR axis and JNK signaling pathways. In conclusion, loganin blocks MPTP-induced neurotoxicity and abnormal development in zebrafish. J. Cell. Biochem. 118: 615-628, 2017. © 2016 Wiley Periodicals, Inc.

Decreased blood hepatitis B surface antibody levels linked to e-waste lead exposure in preschool children.

Lead (Pb) is a widespread environmental contaminant that can profoundly affect the immune system in vaccinated children. To explore the association between blood Pb and HBsAb levels in children chronically exposed to Pb, we measured hepatitis B surface antibody (HBsAb) titers, to reflect the immune response in the children of Guiyu, an electronic and electrical waste (e-waste) recycling area well known for environmental Pb contamination. We performed secondary exploratory analyses of blood Pb levels and plasma HBsAb titers in samples, taken in two phases between 2011 and 2012, from 590 children from Guiyu (exposed group) and Haojiang (reference group). Children living in the exposed area had higher blood Pb levels and lower HBsAb titers compared with children from the reference area. At each phase, generalized linear mixed models (GLMMs) showed that HBsAb titers were significantly negatively associated with child blood Pb levels. This work shows that a decreased immune response to hepatitis B vaccine and immune system might have potential harm to children with chronic Pb exposure. Importantly, nearly 50% of chronically exposed children failed to develop sufficient immunity to hepatitis in response to vaccination. Thus different vaccination strategies are needed for children living under conditions of chronic Pb exposure.

Knockdown of kinesin KIF11 abrogates directed migration in response to epidermal growth factor-mediated chemotaxis.

Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14-45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44-54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.

HMGB1 contributes to regeneration after spinal cord injury in adult zebrafish.

High mobility group box 1 (HMGB1, also called amphoterin) facilitates neurite outgrowth in early development, yet can exacerbate pathology and inhibit regeneration by inducing adverse neuroinflammation when released from dying cells, suggesting that HMGB1 plays a critical, yet undefined role in neuroregeneration. We explored whether HMGB1 contributes to recovery after complete spinal cord transection in adult zebrafish. Quantitative PCR and in situ hybridization revealed that HMGB1 mRNA levels decreased between 12 h to 11 days after spinal cord injury (SCI), then returned to basal levels by 21 days. Western blot and immunohistological analyses indicated that the time course of HMGB1 protein expression after SCI parallels that of mRNA. Immunofluorescence staining revealed that HMGB1 translocates from nuclei into the cytoplasm of spinal motoneurons at 4 and 12 h (acute stage) following SCI, then accumulates in the nuclei of motoneurons during the ensuing chronic stage (after 6 days following SCI). Immunohistology of transgenic zebrafish, expressing green fluorescent protein in blood vessels, showed enhanced HMGB1 expression in blood vessels in the vicinity of motoneurons. Application of anti-sense HMGB1 morpholinos inhibited locomotor recovery by 34 % and decreased axonal regeneration by 34 % compared to fish treated with a control morpholino. The present study shows that HMGB1 expression increases in both endothelial cells and motoneurons, suggesting that HMGB1 promotes recovery from SCI not only through enhancing neuroregeneration, but also by increasing angiogenesis. The inflammatory effects of HMGB1 are minimized through the decrease in HMGB1 expression during the acute stage.

Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1.

Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, ß-actin, α-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.

The DNA-PK catalytic subunit regulates Bax-mediated excitotoxic cell death by Ku70 phosphorylation.

DNA repair deficiency results in neurodegenerative disease and increased susceptibility to excitotoxic cell death, suggesting a critical but undefined role for DNA damage in neurodegeneration. We compared DNA damage, Ku70-Bax interaction, and Bax-dependent excitotoxic cell death in kainic acid-treated primary cortical neurons derived from both wild-type mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wild-type and Prkdc(-/-) neurons, kainic acid treatment resulted in rapid induction of DNA damage (53BP1 foci formation) followed by nuclear pyknosis. Bax deficiency, by either Bax shRNA-mediated knockdown or gene deletion, protected wild-type and heterozygous but not Prkdc(-/-) neurons from kainate-induced excitotoxicity. Cotransfection of DNA-PKcs with Bax shRNA restored Bax shRNA-mediated neuroprotection in Prkdc(-/-) neurons, suggesting that DNA-PKcs is required for kainate-induced activation of the pro-apoptotic Bax pathway. Immunoprecipitation studies revealed that the DNA-PKcs-nonphosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wild-type Ku70, suggesting that DNA-PKcs-mediated Ku70 phosphorylation causes release of Bax from Ku70. In support of this, kainic acid induced translocation of a Bax-EGFP fusion protein to the mitochondria in the presence of a cotransfected wild-type, but not mutant Ku70 (S6A/S51A) gene when examined at 4 and 8 h following kainate addition. We conclude that DNA-PKcs links DNA damage to Bax-dependent excitotoxic cell death, by phosphorylating Ku70 on serines 6 and/or 51, to initiate Bax translocation to the mitochondria and directly activate a pro-apoptotic Bax-dependent death cascade.

Status epilepticus-induced somatostatinergic hilar interneuron degeneration is regulated by striatal enriched protein tyrosine phosphatase.

Excitotoxic cell death is one of the precipitating events in the development of temporal lobe epilepsy. Of particular prominence is the loss of GABAergic hilar neurons. Although the molecular mechanisms responsible for the selective vulnerability of these cells are not well understood, activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway has been implicated in neuroprotective responses to excitotoxicity in other neuronal populations. Here, we report that high levels of the striatal-enriched protein tyrosine phosphatase (STEP), a key regulator of ERK/MAPK signaling, are found in vulnerable somatostatin-immunoreactive hilar interneurons. Under both control conditions and after pilocarpine-induced status epilepticus (SE), ERK/MAPK activation was repressed in STEP-immunoreactive hilar neurons. This contrasts with robust SE-induced ERK/MAPK activation in the granule cell layer of the dentate gyrus, a cell region that does not express STEP. During pilocarpine-induced SE, in vivo disruption of STEP activity allowed activation of the MAPK pathway, leading to immediate-early gene expression and significant rescue from cell death. Thus, STEP increases the sensitivity of neurons to SE-induced excitotoxicity by specifically blocking a latent neuroprotective response initiated by the MAPK pathway. These findings identify a key set of signaling events that render somatostatinergic hilar interneurons vulnerable to SE-induced cell death.

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex.

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.

DNA damage and nonhomologous end joining in excitotoxicity: neuroprotective role of DNA-PKcs in kainic acid-induced seizures.

DNA repair plays a critical, but imprecisely defined role in excitotoxic injury and neuronal survival throughout adulthood. We utilized an excitotoxic injury model to compare the location and phenotype of degenerating neurons in mice (strain 129-C57BL) deficient in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), an enzyme required for nonhomologous end joining (NHEJ). Brains from untreated adult heterozygous and DNA-PKcs null mice displayed comparable cytoarchitecture and undetectable levels of cell death. By day 1, and extending through 4 days following kainic acid-induced seizures, brains from DNA-PKcs null mice showed widespread neurodegeneration that encompassed the entire hippocampal CA1-CA3 pyramidal cell layer, entorhinal cortex, and lateral septum, with relative sparing of the dentate gyrus granule cell layer and hilus, as judged by toluidine blue, Fluoro-Jade B, and terminal dUTP nick end labeling staining. In contrast, seizure-related neurodegeneration in heterozygous littermates was limited to the CA3 region of the hippocampus. NeuN and calbindin staining revealed a selective decrease in the number and density of NeuN-positive neurons in the pyramidal layers of degenerating regions in both heterozygous and DNA-PKcs null mice. To elucidate the mechanisms leading to cell death, we examined an involvement of the p53 pathway, known to be induced by DNA damage. Addition of pifithrin-alpha, a p53 inhibitor, or expression of a dominant-negative p53 rescued neurons from kainate-induced excitotoxic cell death in primary cortical cultures derived from wildtype, DNA-PKcs heterozygous, or DNA-PKcs null neonatal mice. Moreover, pifithrin-alpha prevented kainate-induced loss of mitochondrial membrane potential, dendrite degeneration, and cell death. Results suggest that NHEJ plays a neuroprotective role in excitotoxicity, within the perforant, Schaffer collateral, hippocampal-septal, and temperoammonic pathways, in part by repairing DNA damage that would otherwise result in activation of a p53-dependent apoptotic cascade.

Oxidative damage and defective DNA repair is linked to apoptosis of migrating neurons and progenitors during cerebral cortex development in Ku70-deficient mice.

DNA repair plays a critical, but imprecisely defined role in neuronal survival during cortical neurogenesis. We examined cortical development in mice deficient for the DNA end-joining protein, Ku70. At gestational day 14.5, corresponding to the peak of neurogenesis, the Ku70(-/-) embryonic cerebral cortex displayed 25- to 30-fold more cell death than heterozygous littermates, as judged by DNA breaks, pyknosis and active caspase-3. In Ku70(-/-) embryos only, large clusters of dying neurons were found in the intermediate zone. Cell death declined until P4, when the number of dying cells became comparable to that in heterozygous mice. Two groups of dying cells were evident: a GLAST(+) neural progenitor population in the subventricular and ventricular zones, and a doublecortin(+) immature neuron population in the intermediate zone, the latter exhibiting strong staining for oxidative DNA damage. Antioxidants and lower oxygen tension reduced the high levels of neuronal death in primary cortical cultures derived from Ku70(-/-) mice, but not the low levels of cell death in wildtype cortical cultures. Results indicate migrating cortical neurons undergo oxidative DNA damage, which is normally repaired by non-homologous end joining. Failure to repair oxidative damage triggers a form of apoptosis involving caspase-3 activation.