Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Studies on the structure, function and expression of phospholipase A2

Phospholipase A2 (PLA2) enzymes are widely distributed and the enzymes possess different functions such as a role in digestion and inflammation. PLA2 catalyzes the hydrolysis of the sn-2 acylester bond of phospholipids, yielding a free fatty acid and a lysophospholipid. Arachidonic acid is a precursor molecule for important lipid mediators such as leukotrienes, prostaglandins and thromboxanes, collectively called eicosanoids. The level of free arachidonic acid in the cell is usually low since the majority of arachidonic acid is esterified at the sn-2 position of phospholipids. Mammalian cells contain several different PLA2 enzymes, each capable of releasing arachidonic acid from phospholipids. Furthermore, the produced lysophospholipids can be used for synthesis of platelet-activating factor (PAF). Both eicosanoids and PAF are involved in inflammatory reactions. Atherosclerosis is a disease of large and medium-sized arteries. The athersosclerotic plaque exhibits some features similar to those of chronic inflammation. Furthermore, PLA2 enzymes have been implicated in the pathogenesis of atherosclerosis. Therefore, PLA2 activities and expression of secretory PLA2 (sPLA2), group IIA, and cPLA2 were investigated in normal and atherosclerotic arterial wall by immunohistochemistry. Both healthy and diseased airteries possessed substantial PLA2 activity and expressed sPLA2 group IIA protein. cPLA2 was only detected in atherosclerotic arteries, in regions consisting of inflammatory infiltrate and smooth muscle cells. The expression of cPLA2 in atherosclerotic plaques indicate that this enzyme is associated with the inflammatory component of atherosclerosis. PLA2 has been reported to play a pivotal role in leukotriene synthesis. Leukotrienes are produced by human neutrophils after calcium ionophore A23187 stimulation. However, equine neutrophils, but not eosinophils, require exogenous arachidonic acid for leukotriene synthesis. Studies were undertaken to investigate if equine neutrophils did express an active calcium-dependent PLA2 enzyme. The group IV calcium-dependent cytosolic PLA2 (cPLA2) from equine neutrophils was purified >6500-fold and found to be catalytically active. A possible difference in the degree of phosphorylation of the cPLA2 protein between equine eosinophils and neutrophils was observed. This might explain the inability of equine neutrophils to produce leukotrienes in the absence of exogenous arachidonic acid. In contrast to human myeloid cells, B-lymphocytes are dependent on exogenous arachidonic acid for leukotriene synthesis. However, it has been demonstrated that homogenates of B-lymphocytes possess PLA2 activity. Therefore, we investigated the expression and regulation of PLA2 enzymes in B-lymphocytes. The results demonstrated that a calcium-independent PLA2 (iPLA2) was abundantly expressed in these cells. A full-length cDNA clone encoding the human iPLA2 was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA clone encoded a 806-Alternativelyamino acid protein. This human iPLA2 protein had an additional 54 amino acid insertion not found in rodent forms of the iPLA2 protein. Alternatively spliced variants of the human iPLA2 transcript were observed in cells of both myeloid and lymphoid origin and in muscle cells. The expression of one splice variant was found to be different in immature and mature myeloid cells. The encoded protein from one of these splice variants was found to function as a negative regulator of iPLA2 activity in transfection experiments. Results obtained with human granulocytes suggest that granulocytes possess calcium- independent PLA2 activity. Furthermore, inhibitor studies indicate that iPLA2 is involved in events leading to the release of arachidonic acid destined for leukotriene synthesis in granulocytes. The human iPLA2 gene was identified by computational identification and it was localized to chromosome 22qI3. 1. The gene consists of at least 17 exons and spans more than 69 kb. Based upon the organization of the gene, the observed splice variants of iPLA2 can be explained. Subcellular fractionation studies were performed to investigate the subcellular localization of the native human iPLA2 protein. This form of iPLA2 contains 54 additional amino acids as compared to the soluble iPLA2 enzyme found in several rodents. After subcellular fractionation, human calcium-independent PLA2 activity and iPLA2 protein were found in the membrane fraction. It is suggested that the addition of the 54 amino acids leads to a membrane associated protein. In conclusion, iPLA2 and its splice variants are expressed in various cells such as B-lymphocytes, myeloid cells and muscle cells. Furthermore, multiple enzymes with distinct function and localization are derived from the iPLA2 transcript by alternative splicing

Very long-term outcome of cognitive behavioral therapy for insomnia : one- and ten-year follow-up of a randomized controlled trial

Insomnia is a common and chronic disorder, and cognitive behavioral therapy (CBT) is the recommended treatment. Very long-term follow-ups of CBT are very rare, and this study aimed to investigate if improvements were stable one and ten years after CBT for insomnia (CBT-i). Based on a three-armed randomized controlled trial of bibliotherapeutic CBT-i, participants received an insomnia-specific self-help book and were randomized to therapist guidance, no guidance, or a waitlist receiving unguided treatment after a delay. Six weeks of treatment was given to 133 participants diagnosed with insomnia disorder. After one and ten years, participants were assessed with self-reports and interviews. Improvements were statistically significant and well maintained at one- and ten-year follow-ups. Average Insomnia Severity Index score [95%CI] was 18.3 [17.7-18.8] at baseline, 10.1 [9.3-10.9] at post-treatment, 9.2 [8.4-10.0] at one- and 10.7 [9.6-11.8] at ten-year follow-up, and 64% and 66% of participants no longer fulfilled criteria for an insomnia diagnosis at one and ten years, respectively. Positive effects of CBT were still present after ten years. Insomnia severity remained low, and two-thirds of participants no longer fulfilled criteria for an insomnia diagnosis. This extends previous findings of CBT, further confirming it as the treatment of choice for insomnia

Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but also in underlying mechanisms for memory formation such as hippocampal long-term potentiation. Our aim was to identify small molecules with stimulatory effects on the signaling of two neurotrophins, the nerve growth factor (NGF) and the brain derived neurotrophic factor (BDNF). To identify molecules that could potentiate neurotrophin signaling, 25,000 molecules were screened, which led to the identification of the triazinetrione derivatives ACD855 (Ponazuril) and later on ACD856, as positive allosteric modulators of tropomyosin related kinase (Trk) receptors. ACD855 or ACD856 potentiated the cellular signaling of the neurotrophin receptors with EC50 values of 1.9 and 3.2 or 0.38 and 0.30 µM, respectively, for TrkA or TrkB. ACD855 increased acetylcholine levels in the hippocampus by 40% and facilitated long term potentiation in rat brain slices. The compounds acted as cognitive enhancers in a TrkB-dependent manner in several different behavioral models. Finally, the age-induced cognitive dysfunction in 18-month-old mice could be restored to the same level as found in 2-month-old mice after a single treatment of ACD856. We have identified a novel mechanism to modulate the activity of the Trk-receptors. The identification of the positive allosteric modulators of the Trk-receptors might have implications for the treatment of Alzheimer’s diseases and other diseases characterized by cognitive impairment