The emerging role of circular RNAs in transcriptome regulation.

Circular RNAs (circRNAs) are a recently discovered form of RNA that has been found to regulate mammalian transcription. CircRNAs are covalently closed, single-stranded transcripts produced from precursor mRNA. While initially circRNAs were considered to be splicing artefacts, next-generation RNA sequencing of non-polyadenylated transcriptomes has recently shown that the expression of circRNAs is widespread and over 20% of expressed genes in examined cells and tissues can produce these transcripts. Until now thousands of circRNAs have been discovered in organisms ranging from Drosophila melanogaster to Homo sapiens. Functional studies indicate that these transcripts regulate expression of protein-coding linear transcripts and thus comprise an important component of gene expression regulation. Here we provide a comprehensive overview on the biology of circRNAs, including the expression patterns and function. Moreover, we discuss current methodologies for the discovery and validation of circular transcripts. Finally, perspectives on the utilization of circRNA as molecular markers of complex diseases are presented.

Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina.

Gliosis of retinal Müller glial cells may have both beneficial and detrimental effects on neurons. To investigate the role of purinergic signaling in ischemia-induced reactive gliosis, transient retinal ischemia was evoked by elevation of the intraocular pressure in wild-type (Wt) mice and in mice deficient in the glia-specific nucleotide receptor P2Y1 (P2Y1 receptor-deficient (P2Y1R-KO)). While control retinae of P2Y1R-KO mice displayed reduced cell numbers in the ganglion cell and inner nuclear layers, ischemia induced apoptotic death of cells in all retinal layers in both, Wt and P2Y1R-KO mice, but the damage especially on photoreceptors was more pronounced in retinae of P2Y1R-KO mice. In contrast, gene expression profiling and histological data suggest an increased survival of amacrine cells in the postischemic retina of P2Y1R-KO mice. Interestingly, measuring the ischemia-induced downregulation of inwardly rectifying potassium channel (Kir)-mediated K(+) currents as an indicator, reactive Müller cell gliosis was found to be weaker in P2Y1R-KO (current amplitude decreased by 18%) than in Wt mice (decrease by 68%). The inner retina harbors those neurons generating action potentials, which strongly rely on an intact ion homeostasis. This may explain why especially these cells appear to benefit from the preserved Kir4.1 expression in Müller cells, which should allow them to keep up their function in the context of spatial buffering of potassium. Especially under ischemic conditions, maintenance of this Müller cell function may dampen cytotoxic neuronal hyperexcitation and subsequent neuronal cell loss. In sum, we found that purinergic signaling modulates the gliotic activation pattern of Müller glia and lack of P2Y1 has janus-faced effects. In the end, the differential effects of a disrupted P2Y1 signaling onto neuronal survival in the ischemic retina call the putative therapeutical use of P2Y1-antagonists into question.

Apolipoprotein E allele 4 is not a sufficient or a necessary predictor of the development of Mild Cognitive Impairment.

The presence of Mild Cognitive Impairment (MCI) and of an apolipoprotein E (apoE) varepsilon4 allele both predict the development of Alzheimer's disease. However, the extent to which this allele also predicts the development of MCI is unclear even though MCI is an early transitional stage in the development of Alzheimer's disease. The present study investigates the prevalence of the apoE varepsilon4 allele in incipient MCI. Participants were recruited from the population-based Leipzig Longitudinal Study of the Aged (LEILA75+). All subjects who were initially cognitively healthy, i.e. did not meet MCI criteria described by Petersen [Petersen RC. Mild cognitive impairment. J Intern Med 2004; 256(3): 183-94], and whose apoE status could be determined were followed-up. After 4.5 years, 15.5% of the cognitively healthy target population had developed MCI. The frequencies of the apoE varepsilon4 genotype did not differ between individuals with incipient MCI (12.9%) and individuals who remained cognitively healthy during the study (18.4%, p>0.5). Consequently, the apoE varepsilon4 genotype is not a necessary or sufficient risk factor for MCI. Further studies need to investigate the influence of the whole range of genetic and environmental risk factors on the course of Alzheimer's disease including the initial development of MCI and the later conversion to Alzheimer's disease.

Enhanced matrix degradation after withdrawal of TGF-beta1 triggers hepatocytes from apoptosis to proliferation and regeneration.

TGF-beta1 is a profibrogenic cytokine participating in deposition of extracellular matrix in fibrotic disorders. In liver, its anti-proliferative/apoptotic effect on hepatocytes promotes fibrosis. The tetracycline-controlled double-transgenic TA(LAP-2)/p(tet)TGF-beta1 mouse provides a model for reversible liver fibrosis. In livers of TGF-beta1-expressing mice, hepatocytes showed synchronous apoptosis detected by DNA laddering and active caspase-3 staining that disappeared when expression of transgenic TGF-beta1 was switched off. In these 'off' mice, perisinusoidal liver fibrosis resolved within 21 days accompanied by elevated proliferation of hepatocytes. Here, we have specified the intermediary stages (2-3 days off and 6 days off) in terms of (i) proliferation (by immunohistochemical staining of proliferating cell nuclear antigen and expression of cyclin D1 mRNA) and (ii) extracellular matrix remodelling processes (by measuring mRNA expression of matrix metalloproteinases-2 and -13 (mmp-2 and mmp-13) and tissue inhibitor of matrix metalloproteinases 1 (timp-1) and quantitative morphometric analysis. In summary, we show a rapidly declining timp-1 mRNA level together with lastingly high mmp-2 and mmp-13 mRNA levels after 2-3 days, suggesting that high matrix-degrading potential represents a prerequisite for the markedly enhanced proliferation of hepatocytes in the early stages after switching off transgenic TGF-beta1.

Activated mitogenic signaling induces a process of dedifferentiation in Alzheimer's disease that eventually results in cell death.

Neurodegeneration in Alzheimer's disease (AD) is associated with the appearance of dystrophic neuronal growth profiles that most likely reflect an impairment of neuronal reorganization. This process of morphodysregulation, which eventually goes awry and becomes a disease itself, might be triggered either by a variety of life events that place an additional burden on the plastic capability of the brain or by genetic pertubations that shift the threshold for decompensation. This paper summarizes recent evidence that impairment of the p21ras intracellular signal transduction, which is is mediated by a hierarchy of phosphorylation signals and eventually results in loss of differentiation control and an attempt of neurons to re-enter the cell-cycle, is critically involved in this process. Neurodegeneration might thus be viewed as an alternative effector pathway of those events that in the dividing cell would lead to cellular transformation. This hypothesis might be of heuristic value for the development of a therapeutic strategy.

Regulated secretion of amyloid precursor protein by TrkA receptor stimulation in rat pheochromocytoma-12 cells is mitogen activated protein kinase sensitive.

We have shown recently in the pheochromocytoma PC-12 cell line, that the activation of the high-affinity receptor for nerve growth factor (NGF), tyrosine kinase receptor (TrkA), results in increased secretion of the amyloid precursor protein (APP) into the culture medium. In order to reveal through which TrkA-associated signaling pathway the secretory APP processing is mediated, signaling cascades activated by TrkA stimulation were selectively inhibited under conditions of selective TrkA stimulation via non-NGF mechanisms and APP secretion into the culture medium was followed by Western analysis. Our data demonstrate, that activation of mitogen activated protein (MAP) kinase alone is sufficient to promote APP secretion, whereas inhibition of MAP kinase will reduce APP secretion only when phospholipase Cgamma or phosphatidylinositol 3-kinase are additionally inhibited. This suggests that pharmacological manipulations activating the MAP kinase pathway may result in increased secretory APP processing.

Neurotrophin binding to the p75 neurotrophin receptor is necessary but not sufficient to mediate NGF-effects on APP secretion in PC-12 cells.

In the present study the pheochromocytoma cell line (PC-12) was used as a model system to determine the role of the two neurotrophin receptors in the regulation of amyloid precursor protein (APP) secretion by nerve growth factor (NGF). To stimulate TrkA and/or p75NTR signaling in PC-12 cells, we used NGF, brain-derived neurotrophic factor (BDNF), and NGF in the presence of an excess of BDNF or the monoclonal antibody 192IgG, to block p75NTR binding to NGF. Our results demonstrate that NGF stimulates APP secretion in a dose dependent fashion with maximum effects at 10 ng/ml, known to saturate high-affinity NGF binding sites. Treatment of PC-12 cells with varying concentrations of BDNF, 1-1,000 ng/ml, did not alter APP secretion, suggesting that binding to p75NTR alone is not sufficient to affect APP secretion. When blocking NGF binding to p75NTR with BDNF or 192IgG, on the other hand, NGF effects on APP secretion were abolished. These findings suggest that in cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on APP secretion. Our data are also consistent with a proposed function of the p75NTR in receptor recruitment and "presentation" of NGF to receptors.

The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling.

The increased expression and/or abnormal processing of the amyloid precursor protein (APP) is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of Alzheimer's disease (AD). Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest. The cleavage of the APP by the alpha-secretase within the beta-amyloid sequence generates nonamyloidogenic C-terminal APP fragments and soluble APPs alpha, which has neurotrophic and neuroprotective activities. Proteolytic processing of APP by beta-secretase, on the other hand, exposes the N-terminus of beta-amyloid, which is liberated after gamma-secretase cleavage at the variable amyloid C-terminus. The resulting 39-43 amino acid beta-amyloid may be neurotoxic and disrupt neuronal connectivity after its accumulation in senile plaques. In this review, we discuss evidence derived from in vitro experiments, suggesting that the stimulation of protein kinase C (PKC)-coupled M1/M3 muscarinic acetylcholine receptors increases the nonamyloidogenic, secretory pathway of APP processing. It has also been shown in animal models that under conditions of reduced M1/M3 muscarinic acetylcholine receptor stimulation the secretory pathway of APP processing is inhibited and that constitutive upregulation of M1/M3-associated PKC increases APP secretion. Thus, the cortical cholinergic hypoactivity characteristic of AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased beta-amyloid generation. It has been shown in vitro that nerve growth factor (NGF)-associated signaling also influences the expression and catabolism of APP. Recent experiments with NGF-responsive cells revealed a specific role for the high-affinity NGF receptor, TrkA, in the increases in secretory APP processing and a role for the low-affinity neurotrophin receptor, p75NTR, in the transcriptional regulation of APP. Therefore, treatments with NGF could ameliorate cortical cholinergic dysfunction in AD. These findings may influence the design of therapeutic strategies aimed at stimulating cholinergic function and at increasing nonamyloidogenic APP processing without elevating APP expression.

p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells.

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.

Transforming growth factor-beta reverses deficient expression of type (I) collagen in cultured fibroblasts of a patient with metageria.

Metageria is a generalized form of acrogeria belonging to the group of premature aging syndromes and is characterized by loss of subcutaneous fat, thinning of the dermis, multiple teleangiectasias and mottled hyperpigmentation. The skin changes present suggest that an altered formation of extracellular matrix might be involved in the pathogenesis of this disease. Fibroblasts obtained from the skin of a patient with this disease revealed a marked reduction of type I collagen expression to about 20% of control levels both at the mRNA and protein level. In addition decreased decorin but unchanged type IV collagen and fibronectin mRNA levels were found. Similar although less pronounced changes were observed in fibroblasts obtained from the sister of this patient showing skin changes compatible with acrogeria. To further analyze the deficient expression of type I collagen run on analysis was performed revealing a decrease of transcription of type I collagen. Incubation of the cells with transforming growth factor-beta, a strong inducer of type I collagen and extracellular matrix formation, restored type I collagen expression both at the mRNA and protein level to amounts comparable with normal skin fibroblasts. These results are consistent with a defect in type I collagen transcription that is readily reversed after incubation with transforming growth factor beta. The deficient synthesis of type I collagen and decorin by dermal fibroblasts might thus contribute to an altered formation of the extracellular matrix resulting in the poikilodermic skin changes observed in this patient.

In vivo regulation of amyloid precursor protein secretion in rat neocortex by cholinergic activity.

The proteolytic cleavage of the amyloid precursor protein (APP) has been shown to be modulated through specific muscarinic receptor activation in vitro in both transfected cell lines and native brain slices, whereas a demonstration of receptor-mediated control of APP processing under in vivo conditions is still lacking. To simulate alterations in muscarinic receptor stimulation in vivo, we have (i) specifically reduced the cortical cholinergic innervation in rats using partial immunolesions with 192IgG-saporin, and (ii) restored cholinergic function in lesioned rats by transplantation of nerve growth factor producing fibroblasts. While total APP levels in cortical homogenates were unaffected by cholinergic deafferentation, we observed a significant reduction in the abundance of secreted APP and a concomitant increase in membrane-bound APP. These changes were reversed in immunolesioned rats with nerve growth factor-producing fibroblasts. There was a strong positive correlation between the ratio of secreted APP to membrane-bound APP and the activity of choline acetyltransferase and M1 muscarinic acetylcholine receptor density (measured by [3H]pirenzepine binding) in experimental groups. Additionally, we observed a transient decrease in the ratio of cortical APP transcripts containing the Kunitz protease inhibitor domain (APP 770 and APP 751) versus APP 695 in rats with cholinergic hypoactivity. The data presented suggest that cortical APP processing is under basal forebrain cholinergic control, presumably mediated through M1 muscarinic acetylcholine receptors on cholinoceptive cortical target cells.

Autosomal recessive cutis laxa syndrome. A case report.

Congenital cutis laxa (CCL) is a rare, genetically heterogeneous connective tissue disorder, manifested by loose, hanging skin, giving the appearance of premature aging. We report a 6-year-old female child with autosomal recessive CCL type III, to assess possible correlations between clinical, ultrastructural, cellular and biochemical features. Morphological aberrations of the elastic and collagen tissue, increased collagen I mRNA expression associated with increased protein synthesis and increased collagenase gene expression of the cutis laxa fibroblasts could be established. Our results suggest that CCL is not only a disease of the elastic fibers of the connective tissue but also of the collagen fibers, with a central role of the fibroblast.