PEDF counteracts DL-α-aminoadipate toxicity and rescues gliotoxic damages in RPE-free chicken retinal explants.

Gliotoxic responses complicate human eye diseases, the causes of which often remain obscure. Here, we activated Müller cells (MCs) by the gliotoxin DL-α-aminoadipate (AAA) and assayed possible protective effects by pigment epithelium-derived factor (PEDF) in RPE-free retinal explants of the E6 chick embryo. These models are suited to analyze gliotoxic reactions in vitro, since the avian retina contains only Müller cells (MCs) as glial components, and the RPE-free explants are devoid of a major PEDF source. ChAT- and AChE-immunohistochemistry (IHC) revealed that AAA treatment disrupted the differentiation of cholinergic amacrine cells in the inner plexiform layer. At the applied concentration of 1 mM AAA, apoptosis of MCs was slightly increased, as shown by TUNEL and caspase-3 activity assays. Concomitantly, cell-free gaps emerged in the middle of the retina, where MCs were swollen and amassed glutamine synthetase (shown by GS and Vimentin IHC). AAA treatment strongly activated MCs, as shown by GFAP IHC, and by an increase of stress-related catalase activity. Remarkably, nearly all effects of AAA on MCs were effectively counter-balanced by 50 ng/ml PEDF co-treatment, as also shown by RT-PCR. These findings suggest that supplementation with PEDF can protect the retina against gliotoxic attacks. Further studies should establish whether PEDF similarly protects a gliotoxic human retina.

Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system.

Cholinergic control of bone development and beyond.

There is ample evidence that cholinergic actions affect the health status of bones in vertebrates including man. Nicotine smoking, but also exposure to pesticides or medical drugs point to the significance of cholinergic effects on bone status, as reviewed here in Introduction. Then, we outline processes of endochondral ossification, and review respective cholinergic actions. In Results, we briefly summarize our in vivo and in vitro studies on bone development of chick and mouse [1,2], including (i) expressions of cholinergic components (AChE, BChE, ChAT) in chick embryo, (ii) characterisation of defects during skeletogenesis in prenatal ChE knockout mice, (iii) loss-of-function experiments with beads soaked in cholinergic components and implanted into chicken limb buds, and finally (iv) we use an in vitro mesenchymal 3D-micromass model that mimics cartilage and bone formation, which also had revealed complex crosstalks between cholinergic, radiation and inflammatory mechanisms [3]. In Discussion, we evaluate non-cholinergic actions of cholinesterases during bone formation by considering: (i) how cholinesterases could function in adhesive mechanisms; (ii) whether and how cholinesterases can form bone-regulatory complexes with alkaline phosphatase (ALP) and/or ECM components, which could regulate cell division, migration and adhesion. We conclude that cholinergic actions in bone development are driven mainly by classic cholinergic, but non-neural cycles (e.g., by acetylcholine); in addition, both cholinesterases can exert distinct ACh-independent roles. Considering their tremendous medical impact, these results bring forward novel research directions that deserve to be pursued.

IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling.

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.

Targeting Notch signalling pathway of cancer stem cells.

Cancer stem cells (CSCs) have been defined as cells within tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. CSCs have been increasingly identified in blood cancer, prostate, ovarian, lung, melanoma, pancreatic, colon, brain and many more malignancies. CSCs have slow growth rate and are resistant to chemotherapy and radiotherapy that lead to the failure of traditional current therapy. Eradicating the CSCs and recurrence, is promising aspect for the cure of cancer. The CSCs like any other stem cells activate the signal transduction pathways that involve the development and tissue homeostasis, which include Notch signaling pathway. The new treatment targets these pathway that control stem-cell replication, survival and differentiation that are under development. Notch inhibitors either single or in combination with chemotherapy drugs have been developed to treat cancer and its recurrence. This approach of targeting signaling pathway of CSCs represents a promising future direction for the therapeutic strategy to cure cancer.

Inflammatory effects of TNFα are counteracted by X-ray irradiation and AChE inhibition in mouse micromass cultures.

As a means to analyze anti-inflammatory effects by radiation and/or by cholinergic mechanisms, we found that cultured primary human osteoblasts express most cholinergic components. After X-ray irradiation, their level of acetylcholinesterase (AChE) was strongly elevated. As a 3D model, we cultured mesenchymal stem cells isolated from E11 mouse embryos as micromass nodules, and differentiated them into chondro- and osteoblasts. They were stimulated by 5 or 10 ng/ml of the inflammatory cytokine TNF-α to mimic an inflammatory condition in vitro, before exposure to 2 Gy X-rays. Effects on chondro- and osteoblasts of TNF-α, of X-rays, or both were analysed by Alcian Blue, or Alizarin Red staining, respectively. Acetylcholinesterase (AChE) activity was visualized histochemically. The results showed that treatment with TNF-α affected cartilage and bone formation in vitro, while X-rays reversed the effects of TNF-α. After irradiation, both AChE and alkaline phosphatase (ALP) activities, a marker for bone mineralization, were raised, suggesting that X-rays stimulated cholinergic mechanisms during calcification. Notably, the TNFα-effects on cultures were also counterbalanced after AChE activity was blocked by BW284c51. These findings suggest a complex crosstalk between radiation, cholinergic and inflammatory mechanisms, which could have wide significances, e.g. for understanding rheumatoid arthritis.

Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation.

Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. AChE is expressed in many tissues during development and in mature organisms, as well as in healthy and diseased states. In search for alternative, "non-classical" functions of cholinesterases (ChEs), AChE could either work within the frame of classic cholinergic systems, but in non-neural tissues ("non-synaptic function"), or act non-enzymatically. Here, we review briefly some of the major ideas and advances of this field, and report on some recent progress from our own experimental work, e.g. that (i) non-neural ChEs have pronounced, predominantly enzymatic effects on early embryonic (limb) development in chick and mouse, that (ii) retinal R28 cells of the rat overexpressing synaptic AChE present a significantly decreased cell proliferation, and that (iii) in developing chick retina ACh-synthesizing and ACh-degrading cells originate from the same postmitotic precursor cells, which later form two locally opposing cell populations. We suggest that such distinct distributions of ChAT(+) vs. AChE(+) cells in the inner half retina provide graded distributions of ACh, which can direct cell differentiation and network formation. Thus, as corroborated by works from many labs, AChE can be considered a highly co-opting protein, which can combine enzymatic and non-enzymatic functions within one molecule.

Intricate paths of cells and networks becoming "Cholinergic" in the embryonic chicken retina.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are the decisive enzymatic activities regulating the availability of acetylcholine (ACh) at a given synaptic or nonsynaptic locus. The only cholinergic cells of the mature inner retina are the so-called starburst amacrine cells (SACs). A type-I SAC, found at the outer border of the inner plexiform layer (IPL), forms a synaptic subband "a" within the IPL, while a type-II SAC located at the inner IPL border projects into subband "d." Applying immunohistochemistry for ChAT and AChE on sections of the chicken retina, we here have revealed intricate relationships of how retinal networks became dominated by AChE or by ChAT reactivities. AChE+ cells were first detectable in an embryonic day (E)4 retina, while ChAT appeared 1 day later in the very same cells; at this stage all are Brn3a+, a marker for ganglion cells (GCs). On either side of a faint AChE+ band, indicating the future IPL, pairs of ChAT+ /AChE- /Brn3a- cells appeared between E7/8. Type-I cells had increased ChAT and lost AChE; type-II cells presented less ChAT, but some AChE on their surfaces. Direct neighbors of SACs tended to express much AChE. Along with maturation, subband "a" presented more ChAT but less AChE; in subband "d" this pattern was reversed. In conclusion, the two retinal cholinergic networks segregate out from one cell pool, become locally opposed to each other, and become dominated by either synthesis or degradation of ACh. These "cholinergic developmental divergences" may also have significant physiologic consequences.