Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

<p>Abstract</p> <p>Background</p> <p>Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied.</p> <p>Methods</p> <p>In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses.</p> <p>Results</p> <p>Under normoxic conditions, a half maximal inhibitory concentration (IC₅₀) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions.</p> <p>Conclusion</p> <p>Our results suggest that BA is capable of improving the effects of tumor therapy in human malignant glioma cells, particularly under hypoxic conditions. Further investigations are necessary to characterize its potential as a radiosensitizer.</p

Protein tyrosine phosphatases and neural development

Das retino-colliculare System dient als gut verstandenes Model, um die molekularen Mechanismen, die der Entwicklung des ZNS zugrunde liegen, zu erforschen. In diesem Zusammenhang steht, dass Mitglieder der Proteintyrosinphosphatase (PTP) Familie während der neuralen Entwicklung verschiedene zelluläre Prozesse, wie Proliferation und Differenzierung, zu steuern scheinen. In der vorliegenden Arbeit liegt der Fokus auf der Untersuchung von PTPs während der retino-collicularen Entwicklung, wobei (1) die klonale Identifizierung, (2) das Expressionsmuster und (3) die funktionelle Bedeutung von PTPs genauer betrachtet wurden. Zu den Ergebnissen der Arbeit zählt, dass sich PTP-Meg2-defiziente Mäuse phänotypisch durch eine hypozelluläre Neuroretina, eine reduzierte Proliferation und durch eine veränderte Differenzierung retinaler Vorläuferzellen auszeichnen. Mittels "Microarray"-Analysen und $\textit {in vivo}$ Tonometrie konnten außerdem Hinweise auf einen glaukomatösen Phänotyp erbracht werden

Tenascins in retinal and optic nerve neurodegeneration

Tenascins represent key constituents of the extracellular matrix (ECM) with major impact on central nervous system (CNS) development. In this regard, several studies indicate that they play a crucial role in axonal growth and guidance, synaptogenesis and boundary formation. These functions are not only important during development, but also for regeneration under several pathological conditions. Additionally, tenascin-C (Tnc) represents a key modulator of the immune system and inflammatory processes. In the present review article, we focus on the function of Tnc and tenascin-R (Tnr) in the diseased CNS, specifically after retinal and optic nerve damage and degeneration. We summarize the current view on both tenascins in diseases such as glaucoma, retinal ischemia, age-related macular degeneration (AMD) or diabetic retinopathy. In this context, we discuss their expression profile, possible functional relevance, remodeling of the interacting matrisome and tenascin receptors, especially under pathological conditions

Extracellular matrix remodeling in the retina and optic nerve of a novel glaucoma mouse model

Glaucoma is a neurodegenerative disease that is characterized by the loss of retinal ganglion cells (RGC) and optic nerve fibers. Increased age and intraocular pressure (IOP) elevation are the main risk factors for developing glaucoma. Mice that are heterozygous (HET) for the mega-karyocyte protein tyrosine phosphatase 2 (PTP-Meg2) show chronic and progressive IOP elevation, severe RGCs loss, and optic nerve damage, and represent a valuable model for IOP-dependent primary open-angle glaucoma (POAG). Previously, evidence accumulated suggesting that glaucomatous neurodegeneration is associated with the extensive remodeling of extracellular matrix (ECM) molecules. Unfortunately, little is known about the exact ECM changes in the glaucomatous retina and optic nerve. Hence, the goal of the present study was to comparatively explore ECM alterations in glaucomatous PTP-Meg2 HET and control wild type (WT) mice. Due to their potential relevance in glaucomatous neurodegeneration, we specifically analyzed the expression pattern of the ECM glycoproteins fibronectin, laminin, tenascin-C, and tenascin-R as well as the proteoglycans aggrecan, brevican, and members of the receptor protein tyrosine phosphatase beta/zeta (RPTP$\beta$/$\zeta$) family. The analyses were carried out in the retina and optic nerve of glaucomatous PTP-Meg2 HET and WT mice using quantitative real-time PCR (RT-qPCR), immunohistochemistry, and Western blot. Interestingly, we observed increased fibronectin and laminin levels in the glaucomatous HET retina and optic nerve compared to the WT group. RT-qPCR analyses of the laminins $\alpha$4, $\beta$2 and $\gamma$3 showed an altered isoform-specific regulation in the HET retina and optic nerve. In addition, an upregulation of tenascin-C and its interaction partner RPTP$\beta$/$\zeta$/phosphacan was found in glaucomatous tissue. However, comparable protein and mRNA levels for tenascin-R as well as aggrecan and brevican were observed in both groups. Overall, our study showed a remodeling of various ECM components in the glaucomatous retina and optic nerve of PTP-Meg2 HET mice. This dysregulation could be responsible for pathological processes such as neovascularization, inflammation, and reactive gliosis in glaucomatous neurodegeneration

Loss of the extracellular matrix molecule tenascin-C leads to absence of reactive gliosis and promotes anti-inflammatory cytokine expression in an autoimmune glaucoma mouse model

Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc). Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. Ten weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells, and the expression of different cytokines in retina and optic nerve tissue in all four groups. IOP and retinal function were comparable in all groups. Although RGC loss was less severe in KO ONA, WT as well as KO mice displayed a significant cell loss after immunization. Compared to KO ONA, less $\beta$III-$tubulin^{+}$ axons, and downregulated oligodendrocyte markers were noted in WT ONA optic nerves. In retina and optic nerve, we found an enhanced $GFAP^{+}$ staining area of astrocytes in immunized WT. A significantly higher number of retinal $Iba1^{+}$ microglia was found in WT ONA, while a lower number of $Iba1^{+}$ cells was observed in KO ONA. Furthermore, an increased expression of the glial markers $\textit {Gfap, Iba1, Nos2,}$ and $\it Cd68$ was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA. In addition, pro-inflammatory $\it Tnfa$ expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory $\it Tgfb1$ expression was measured in KO ONA animals. We conclude that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model for the first time offers the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling

Brevican, Neurocan, Tenascin-C, and Tenascin-R act as important regulators of the interplay between perineuronal nets, synaptic integrity, inhibitory interneurons, and Otx2

Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix (ECM) structure called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons, and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C, and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. In addition, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Furthermore, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs, did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin interneuron and PNN maturation, and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C, and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution, and Otx2 in the V1

Brain volume increase and neuronal plasticity underly predator-induced morphological defense expression in Daphnia longicephala\textit {Daphnia longicephala}

Predator-induced phenotypic plasticity describes the ability of prey to respond to an increased predation risk by developing adaptive phenotypes. Upon the perception of chemical predator cues, the freshwater crustacean $\textit {Daphnia longicephala}$ develops defensive crests against its predator $\it Notonecta$ spec. (Heteroptera). Chemical predator perception initiates a cascade of biological reactions that leads to the development of these morphological features. Neuronal signaling is a central component in this series, however how the nervous system perceives and integrates environmental signals is not well understood. As neuronal activity is often accompanied by functional and structural plasticity of the nervous system, we hypothesized that predator perception is associated with structural and functional changes of nervous tissues. We observe structural plasticity as a volume increase of the central brain, which is independent of the total number of brain cells. In addition, we find functional plasticity in form of an increased number of inhibitory post-synaptic sites during the initial stage of defense development. Our results indicate a structural rewiring of nerve-cell connections upon predator perception and provide important insights into how the nervous system of prey species interprets predator cues and develops cost–benefit optimized defenses

Simultaneous complement response via lectin pathway in retina and optic nerve in an experimental autoimmune glaucoma model

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride ($\it n$ = 5–6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more $\it C3$ mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients

S100B immunization triggers NFκ\kappaB and complement activation in an autoimmune glaucoma model

In glaucoma, latest studies revealed an involvement of the complement system with and without an elevated intraocular pressure. In the experimental autoimmune glaucoma model, immunization with antigens, such as S100B, lead to retinal ganglion cell (RGC) loss and optic nerve degeneration after 28 days. Here, we investigated the timeline of progression of the complement system, toll-like-receptor 4 (TLR4), and the transcription factor nucleus factor-kappa B (NF$\kappa$B). Therefore, rats were immunized with S100B protein (S100) and analyzed at 3, 7, and 14 days. RGC numbers were comparable at all points in time, whereas a destruction of S100 optic nerves was noted at 14 days. A significant increase of mannose binding lectin (MBL) was observed in S100 retinas at 3 days. Subsequently, significantly more MBL$^{+}$ cells were seen in S100 optic nerves at 7 and 14 days. Accordingly, C3 was upregulated in S100 retinas at 14 days. An increase of interleukin-1 beta was noted in S100 aqueous humor samples at 7 days. In this study, activation of complement system via the lectin pathway was obvious. However, no TLR4 alterations were noted in S100 retinas and optic nerves. Interestingly, a significant NF$\kappa$B increase was observed in S100 retinas at 7 and 14 days. We assume that NF$\kappa$B activation might be triggered via MBL leading to glaucomatous damage

Transfer of the experimental autoimmune glaucoma model from rats to mice

Studies have suggested an involvement of the immune system in glaucoma. Hence, a rat experimental autoimmune glaucoma model (EAG) was developed to investigate the role of the immune response. Here, we transferred this model into mice. Either 0.8 mg/mL of the optic nerve antigen homogenate (ONA; ONA 0.8) or 1.0 mg/mL ONA (ONA 1.0) were injected in 129/Sv mice. Controls received sodium chloride. Before and 6 weeks after immunization, the intraocular pressure (IOP) was measured. At 6 weeks, retinal neurons, glia cells, and synapses were analyzed via immunohistology and quantitative real-time PCR (RT-qPCR). Additionally, optic nerves were examined. The IOP stayed in the normal physiological range throughout the study ($\it p$ > 0.05). A significant reduction of retinal ganglion cells (RGCs) was noted in both immunized groups ($\it p$ < 0.001). Remodeling of glutamatergic and GABAergic synapses was seen in ONA 1.0 retinas. Furthermore, both ONA groups revealed optic nerve degeneration and macrogliosis (all: $\it p$ < 0.001). An increase of activated microglia was noted in ONA retinas and optic nerves ($\it p$ < 0.05). Both ONA concentrations led to RGC loss and optic nerve degeneration. Therefore, the EAG model was successfully transferred from rats to mice. In further studies, transgenic knockout mice can be used to investigate the pathomechanisms of glaucoma more precisely