GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis.

Searching for chemical agents and molecular targets protecting against secondary neuronal damage reflects one major issue in neuroscience. Cannabinoids limit neurodegeneration by activation of neuronal G protein-coupled cannabinoid receptor 1 (CB1 ) and microglial G protein-coupled cannabinoid receptor 2 (CB2 ). However, pharmacological experiments with CB1 /CB2 -deficient mice unraveled the existence of further, so-called non-CB1 /non-CB2 G protein-coupled receptor (GPR) subtypes. GPR55, whose function in the brain is still poorly understood, represents a novel target for various cannabinoids. Here, we investigated whether GPR55 reflects a potential beneficial target in neurodegeneration by using the excitotoxicity in vitro model of rat organotypic hippocampal slice cultures (OHSC). l-α-Lysophosphatidylinositol (LPI), so far representing the most selective agonist for GPR55, protected dentate gyrus granule cells and reduced the number of activated microglia after NMDA (50 µM) induced lesions. The relevance of GPR55 activation for LPI-mediated neuroprotection was determined by using Gpr55 siRNA. Microglia seems to mediate the observed neuroprotection since their depletion in OHSC attenuated the beneficial effects of LPI. Moreover, LPI alone induced microglia chemotaxis but conversely significantly attenuated ATP triggered microglia migration. These effects seemed to be independent from intracellular Ca(2+) and p38 or p44/p42 MAPK phosphorylation. In conclusion, this study unmasked a yet unknown role for GPR55 in neuroprotection driven by LPI-mediated modulation of microglia function.

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Gastric and esophagogastric junction cancers are heterogeneous and aggressive tumors with an unpredictable response to cytotoxic treatment. New methods allowing for the analysis of drug resistance are needed. Here, we describe a novel technique by which human tumor specimens can be cultured ex vivo, preserving parts of the natural cancer microenvironment. Using a tissue chopper, fresh surgical tissue samples were cut in 400 μm slices and cultivated in 6‐well plates for up to 6 days. The slices were processed for routine histopathology and immunohistochemistry. Cytokeratin stains (CK8, AE1/3) were applied for determining tumor cellularity, Ki‐67 for proliferation, and cleaved caspase‐3 staining for apoptosis. The slices were analyzed under naive conditions and following 2–4 days in vitro exposure to 5‐FU and cisplatin. The slice culture technology allowed for a good preservation of tissue morphology and tumor cell integrity during the culture period. After chemotherapy exposure, a loss of tumor cellularity and an increase in apoptosis were observed. Drug sensitivity of the tumors could be assessed. Organotypic slice cultures of gastric and esophagogastric junction cancers were successfully established. Cytotoxic drug effects could be monitored. They may be used to examine mechanisms of drug resistance in human tissue and may provide a unique and powerful ex vivo platform for the prediction of treatment response.

OBJECTIVE: Leptin-deficient (ob/ob) mice are obese and infertile. Dysfunctions of the ovaries are preferentially related to leptin-deficiency. DESIGN: Morphological and molecular biological obesity-dependent changes in ob/ob ovaries. SUBJECTS: Ovaries were obtained from three-month-old mice either homozygote (ob/ob) and heterozygote (ob/ þ ) or wild-type (C57BL6, WT) for the investigation by light and electron microscopy, as well as for western blot analysis of lectin-like oxidised low density lipoprotein receptor (LOX-1), Toll-like receptor 4 (TLR4), CD36, cleaved caspase-3, microtubule-associated protein light chain 3 (LC3), and the steroidogenic acute regulatory protein (StAR). RESULTS: Compared with control ovaries with corpora lutea, ob/ob ovaries lacked corpora lutea, follicular atresia was at a higher rate; lipid droplets accumulated in follicle cells and in the oocyte with damaged mitochondria; the basement membrane of follicles was thickened. LOX-1 and CD36 expressions were comparable for all three groups. Ob/ob ovaries showed significantly higher levels of TLR4 and cleaved caspase-3 than the ones from the control groups. The high LC3-II/I ratio in the WT and ob/ þ ovaries was related to the presence of corpora lutea. The StAR protein was lower in the ob/ob ovaries signifying reduced steroidogenesis. CONCLUSIONS: Excessive lipid storage causes disorders of ovarian function in ob/ob mice. The local lipid overload leads to advanced follicular atresia with apoptosis and defect steroidogenesis. We suggest that the changes in lipid metabolism lead to increased oxidative stress and thereby, they are an important reason of anovulation and infertility.

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BackgroundCannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.Design and MethodsWe examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated.ResultsA predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.ConclusionsThe present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

The non-viral delivery of small RNA molecules like siRNAs still poses a major bottleneck for their successful application in vivo. This is particularly true with regard to crossing physiological barriers upon systemic administration. We have previously established polyethylenimine (PEI)-based complexes for therapeutic RNA formulation. These nanoplexes mediate full RNA protection against nucleolytic degradation, delivery to target tissues as well as cellular uptake, intracellular release and therapeutic efficacy in preclinical in vivo models. We herein present data on different polyplex modifications for the defined improvement of physicochemical and biological nanoparticle properties and for targeted delivery. (i) By non-covalent modifications of PEI polyplexes with phospholipid liposomes, ternary complexes ("lipopolyplexes") are obtained that combine the favorable features of PEI and lipid systems. Decreased cytotoxicity and highly efficient delivery of siRNA is achieved. Some lipopolyplexes also allow prolonged storage, thus providing formulations with higher stability. (ii) Novel tyrosine modifications of low molecular weight PEI offer further improvement of stability, biocompatibility, and knockdown efficacy of resulting nanoparticles. (iii) For ligand-mediated uptake, the shielding of surface charges is a critical requirement. This is achieved by PEI grafting with polyethylene glycol (PEG), prior to covalent coupling of anti-HER1 antibodies (Erbitux®) as ligand for targeted delivery and uptake. Beyond tumor cell culture, analyses are extended towards tumor slice cultures from tumor xenograft tissues which reflect more realistically the in vivo situation. The determination of siRNA-mediated knockdown of endogenous target genes, i.e., the oncogenic survival factor survivin and the oncogenic receptor tyrosine kinase HER2, reveals nanoparticle penetration and biological efficacy also under intact tissue and stroma conditions.