Faramarz Dehghani

Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin

In mammals, many daily cycles are driven by a central circadian clock, which is based on the cell-autonomous rhythmic expression of clock genes. It is not clear, however, how peripheral cells are able to interpret the rhythmic signals disseminated from this central oscillator. Here we show that cycling expression of the clock gene Period1 in rodent pituitary cells depends on the heterologous sensitization of the adenosine A2b receptor, which occurs through the nocturnal activation of melatonin mt1 receptors. Eliminating the impact of the neurohormone melatonin simultaneously suppresses the expression of Period1 and evokes an increase in the release of pituitary prolactin. Our findings expose a mechanism by which two convergent signals interact within a temporal dimension to establish high-amplitude, precise and robust cycles of gene expression.

Prognostic implication of histopathological, immunohistochemical and clinical features of oligodendrogliomas: a study of 89 cases

Abstract Histopathological, immunohistochemical and clinical parameters were correlated with survival in 89 cases of oligodendroglioma (65 patients with grade II and 24 patients with grade III of the WHO classification). Median survival time and 5-year survival rate were 3.5 years and 76% for patients with oligodendroglioma grade II and 0.875 years and 23% for patients with oligodendroglioma grade III. The tumor biopsy specimens were immunohistochemically analyzed for Ki 67 (MIB-1), vimentin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) and synaptophysin. MIB-1 nuclear labeling index ranged from 0.0% to 33.4%; vimentin-immunoreactive tumor cells were found in 25 cases. MIB-1 nuclear labeling index and vimentin immunoreaction showed a significant statistical correlation to the 5-year survival rate of the patients. Tumors with vimentin expression (n = 25) and/ or high MIB-1 labeling index (n = 26) had a poorer prognosis than tumors lacking vimentin expression (n = 57) and/or displaying a low MIB-1 labeling index (n = 56). The expression of immunoreactivity for GFAP (n = 53), NSE (n = 23) and synaptophysin (n = 15) appeared to be of no prognostic relevance. Patients with gross total tumor resection (n = 47) had a median survival time and 5-year survival rate of 3.3 years and 84% compared to 1.2 years and 42% for patients with subtotal resection (n = 41). The comparison between patients who underwent surgery alone (n = 53) and those who had surgery plus postoperative radiation therapy showed no significant survival benefit from postoperative radiation therapy. In conclusion, tumor grade, MIB-1 labeling index, expression of vimentin and the extent of surgery are shown to be of prognostic relevance for patients with oligodendroglioma.

Temporal dynamics of mouse hippocampal clock gene expression support memory processing

Hippocampal plasticity and mnemonic processing exhibit a striking time‐of‐day dependence and likely implicate a temporally structured replay of memory traces. Molecular mechanisms fulfilling the requirements of sensing time and capturing time‐related information are coded in dynamics of so‐called clock genes and their protein products, first discovered and described in the hypothalamic suprachiasmatic nucleus. Using real‐time PCR and immunohistochemical analyses, we show that in wildtype mice core clock components (mPer1/PER1, mPer2/PER2, mCry1/CRY1, mCry2/CRY2, mClock/CLOCK, mBmal1/BMAL1) are expressed in neurons of all subregions of the hippocampus in a time‐locked fashion over a 24‐h (diurnal) day/night cycle. Temporal profiling of these transcriptional regulators reveals distinct and parallel peaks, at times when memory traces are usually formed and/or consolidated. The coordinated rhythmic expression of hippocampal clock gene expression is greatly disordered in mice deficient for the clock gene mPer1, a key player implicated in both, maintenance and adaptative plasticity of circadian clocks. Moreover, Per1‐knockout animals are severely handicapped in a hippocampus‐dependent long‐term spatial learning paradigm. We propose that the dynamics of hippocampal clock gene expression imprint a temporal structure on memory processing and shape at the same time the efficacy of behavioral learning.

Transcription Factors in Neuroendocrine Regulation: Rhythmic Changes in pCREB and ICER Levels Frame Melatonin Synthesis

Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element–binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes thein vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis.

Amyloid β-protein (Aβ)-containing astrocytes are located preferentially near N-terminal-truncated Aβ deposits in the human entorhinal cortex

Abstract The deposition of the amyloid β-protein (Aβ) is a pathological hallmark of Alzheimer’s disease (AD). Aβ is a peptide consisting of 39–43 amino acids and is derived by β- and γ-secretase cleavage from the Aβ protein precursor (AβPP). An N-terminal-truncated form of Aβ can occur following α- and γ-secretase cleavage of AβPP. Fleecy amyloid is a recently identified distinct type of Aβ deposits occurring in the internal layers (pri-α, pri-β and pri-γ) of the human entorhinal cortex. Fleecy amyloid consists exclusively of N-terminal-truncated Aβ and is a transient form of Aβ deposits, which disappears in late-stage β-amyloidosis. In this study, the entorhinal cortex of 15 cases with AD-related pathology was used to examine astrocytes in the vicinity of N-terminal-truncated Aβ in fleecy amyloid of the layers pri-α, pri-β, and pri-γ in comparison to astrocytes in the vicinity of full-length Aβ in layers pre-β and pre-γ. Immunohistochemistry was performed with antibodies directed against AβPP, Aβ40, Aβ42, Aβ17–24, Aβ1–17 and Aβ8–17 as well as by double-labeling with antibodies directed against Aβ17–24, Aβ42, and glial fibrillary acid protein (GFAP). A large number of GFAP-positive astrocytes containing N-terminal-truncated Aβ fragments appeared in the vicinity of N-terminal-truncated Aβ, whereas Aβ-containing astrocytes were rarely seen in the vicinity of full-length Aβ. These results suggest that N-terminal-truncated Aβ peptide may be cleared preferentially from the extracellular space by astrocytic uptake and processing. Such an astroglial uptake of N-terminal-truncated Aβ may account for the transient nature of fleecy amyloid and point to the use of N-terminal truncation of Aβ in potential therapeutic strategies aimed at preventing the brain from amassing full-length Aβ deposits.

Microglia promote colonization of brain tissue by breast cancer cells in a Wnt-dependent way.

Although there is increasing evidence that blood‐derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf‐2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2‐like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor‐educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy.

Interleukin-1β exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures

The effects of interleukin (IL)‐1β and IL‐1 receptor antagonist (IL‐1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N‐methyl‐d‐aspartate (NMDA) and treated with IL‐1β (6 ng/mL) or IL‐1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL‐1β did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA‐lesioned OHSCs with IL‐1β exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA‐lesioned cultures with IL‐1ra significantly attenuated NMDA‐induced neuronal damage and reduced the number of microglial cells, whereas application of IL‐1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL‐1β directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL‐1β induces microglial activation but is not neurotoxic per se; (iii) IL‐1β enhances excitotoxic neuronal damage and microglial activation and (iv) IL‐1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.

2‐Arachidonoylglycerol elicits neuroprotective effects on excitotoxically lesioned dentate gyrus granule cells via abnormal‐cannabidiol‐sensitive receptors on microglial cells

Endocannabinoids like 2‐arachidonoylglycerol (2‐AG) exert neuroprotective effects after brain injuries. According to current concepts, these neuroprotective effects are due to interactions between 2‐AG and cannabinoid (CB)1 receptors on neurons. Moreover, 2‐AG modulates migration and proliferation of microglial cells which are rapidly activated after brain lesion. This effect is mediated via CB2‐ and abnormal‐cannabidiol (abn‐CBD)‐sensitive receptors. In the present study, we investigated whether the abn‐CBD‐sensitive receptor on microglial cells contributes to 2‐AG‐mediated neuroprotection in organotypic hippocampal slice cultures (OHSCs) after excitotoxic lesion induced by NMDA (50 μM) application for 4 h. This lesion caused neuronal damage and accumulation of microglial cells within the granule cell layer. To analyze the role of abn‐CBD‐sensitive receptors for neuroprotection and microglial cell accumulation, two agonists of the abn‐CBD‐sensitive receptor, abn‐CBD or 2‐AG, two antagonists, 1,3‐dimethoxy‐5‐methyl‐2‐[(1R,6R)‐3‐methyl‐6‐(1‐methylethenyl)‐2‐cyclohexen1‐yl]‐benzene (O‐1918) or cannabidiol (CBD), and the CB1 receptor antagonist AM251, were applied to NMDA‐lesioned OHSC. Propidium iodide (PI) labeling was used as a marker of degenerating neurons and isolectin B4 (IB4) as a marker of microglial cells. Application of both, abn‐CBD or 2‐AG to lesioned OHSC significantly decreased the number of IB  4+ microglial cells and PI+ neurons in the dentate gyrus. In contrast to AM251, application of O‐1918 or CBD antagonized these effects. When microglial cells were depleted by preincubation of OHSC with the bisphosphonate clodronate (100 μg/mL) for 5 days before excitotoxic lesion, 2‐AG and abn‐CBD lost their neuroprotective effects. We therefore propose that the endocannabinoid 2‐AG exerts its neuroprotective effects via activation of abn‐CBD‐sensitive receptors on microglial cells.

R-Flurbiprofen Reduces Neuropathic Pain in Rodents by Restoring Endogenous Cannabinoids

Background R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPARγ and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.

Interleukin-4, interleukin-10, and interleukin-1-receptor antagonist but not transforming growth factor-β induce ramification and reduce adhesion molecule expression of rat microglial cells

The activity of microglial cells is strictly controlled in order to maintain central nervous system (CNS) immune privilege. We hypothesized that several immunomodulatory factors present in the CNS parenchyma, i.e., the Th2‐derived cytokines interleukin (IL)‐4 and IL‐10, interleukin‐1‐receptor‐antagonist (IL‐1‐ra), or transforming growth factor (TGF)‐β can modulate microglial morphology and functions. Microglial cells were incubated with IL‐4, IL‐10, IL‐1‐ra, TGF‐β, or with astrocyte conditioned media (ACM) and were analyzed for morphological changes, expression of intercellular adhesion molecule (ICAM)‐1, and secretion of IL‐1β or tumor necrosis factor (TNF)‐α. Whereas untreated controls showed an amoeboid morphology both Th2‐derived cytokines, IL‐1‐ra, and ACM induced a morphological transformation to the ramified phenotype. In contrast, TGF‐β‐treated microglial cells showed an amoeboid morphology. Even combined with the neutralizing antibodies against IL‐4, IL‐10, or TGF‐β ACM induced microglial ramification. Furthermore, ACM did not contain relevant amounts of IL‐4 and IL‐10, as measured by enzyme‐linked immunosorbent assay (ELISA). Flow cytometry showed that lipopolysaccharide (LPS)‐induced ICAM‐1‐expression on microglial cells was strongly suppressed by ACM, significantly modulated by IL‐4, IL‐10, or IL‐1‐ra, but not influenced by TGF‐β. The LPS‐induced secretion of IL‐1β and TNF‐α was only reduced after application of ACM, whereas IL‐4 or IL‐10 did not inhibit IL‐1β‐ or TNF‐α secretion. TGF‐β enhanced IL‐1β‐ but not TNF‐α secretion. In summary, we demonstrate that IL‐4, IL‐10, and IL‐1‐ra induce microglial ramification and reduce ICAM‐1‐expression, whereas the secretion of proinflammatory cytokines is not prevented. TGF‐β has no modulating effects. Importantly, unidentified astrocytic factors that are not identical with IL‐4, IL‐10, or TGF‐β possess strong immunomodulatory properties.