Citicoline (CDP‐choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke

CDP‐choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP‐choline and placebo groups, CDP‐choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t‐PA. Several mechanisms have been proposed to explain the beneficial actions of CDP‐choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP‐choline. Fischer rats and Sirt1^?/? mice were subjected to permanent focal ischemia. CDP‐choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP‐choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP‐choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP‐choline. CDP‐choline failed to reduce infarct volume in Sirt1^?/? mice. Our present results demonstrate a robust effect of CDP‐choline like SIRT1 activator by up‐regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke.

     

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used ¹⁵N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before ¹H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.     

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-¹H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.     

Measurement of Free Choline in Plant Leaves by Capillary Electrophoresis

A low pH capillary electrophoresis (CE) was used for the measurement of free choline in plant leaves. Choline in the leaf extract was first converted to the benzoyl ester and put into CE. A well-resolved peak in the electropherogram was easily obtained. Involvement of enzymes in a two-step oxidation of choline to glycine betaine was evaluated in different plant species with the same mehod developed for glycine betaine and betaine aldehyde.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Lifelong choline supplementation ameliorates Alzheimer’s disease pathology and associated cognitive deficits by attenuating microglia activation

Currently, there are no effective therapies to ameliorate the pathological progression of Alzheimer's disease (AD). Evidence suggests that environmental factors may contribute to AD. Notably, dietary nutrients are suggested to play a key role in mediating mechanisms associated with brain function. Choline is a B‐like vitamin nutrient found in common foods that is important in various cell functions. It serves as a methyl donor and as a precursor for production of cell membranes. Choline is also the precursor for acetylcholine, a neurotransmitter which activates the alpha7 nicotinic acetylcholine receptor (α7nAchR), and also acts as an agonist for the Sigma‐1 R (σ1R). These receptors regulate CNS immune response, and their dysregulation contributes to AD pathogenesis. Here, we tested whether dietary choline supplementation throughout life reduces AD‐like pathology and rescues memory deficits in the APP/PS1 mouse model of AD. We exposed female APP/PS1 and NonTg mice to either a control choline (1.1 g/kg choline chloride) or a choline‐supplemented diet (5.0 g/kg choline chloride) from 2.5 to 10 months of age. Mice were tested in the Morris water maze to assess spatial memory followed by neuropathological evaluation. Lifelong choline supplementation significantly reduced amyloid‐β plaque load and improved spatial memory in APP/PS1 mice. Mechanistically, these changes were linked to a decrease of the amyloidogenic processing of APP, reductions in disease‐associated microglial activation, and a downregulation of the α7nAch and σ1 receptors. Our results demonstrate that lifelong choline supplementation produces profound benefits and suggest that simply modifying diet throughout life may reduce AD pathology.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

辽宁碱蓬胆碱单加氧酶(CMO)基因启动子分离及功能元件分析

根据已知的辽宁碱蓬CMO cDNA 5′端序列设计两个基因特异的反向引物(CR1,CR2),通过衔接头PCR获得了CMO基因起始密码子上游498 bp的序列。根据所获得的序列设计两个基因特异的反向引物(CR3,CR4),用CR2、CR3、CR4分别与4个简并引物配对,通过TAIL-PCR扩增,获得了约2 kb的序列。经Sequencer软件拼接上述两段序列,获得了CMO基因起始密码子上游2,332 bp的序列。用TSSP-TCM软件分析此序列,预测出转录起始点(C)位于起始密码子上游128 bp处,由此我们获得了2,204 bp的SlCMO启动子序列。用PLACE软件分析此序列,发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如盐诱导元件GAAAAA,冷胁迫诱导元件CANNTG,ABA 响应因子NAACAA,水胁迫元件CGGTTG和伤害诱导元件GTTAGGTTC等,是一个强的胁迫诱导启动子。辽宁碱蓬胆碱单加氧酶基因盐诱导启动子的获得,为盐诱导启动子功能元件分析提供了可能,为进一步研究启动子结构与功能的相互关系、CMO基因的表达调控机制奠定了基础。     

l-Theanine and NEP1-40 promote nerve regeneration and functional recovery after brachial plexus root avulsion

Brachial plexus root avulsion causes severe sequelae Treatments and prognosis face many problems, including inflammatory reaction, oxidative damage, and myelin related inhibitory effect. l-Theanine has anti-inflammatory, anti-oxidative, and neuroprotective effects. NEP1-40 competitively inhibits Nogo-66 receptor (NgR1) promotes axonal regeneration. Forty-eight Sprague-Dawley rats were randomly assigned into four groups to establish an animal model of brachial plexus root avulsion. Inflammation and oxidative damage were evaluated by spectrophotometry and motor function of the upper limbs was assessed via Terzis grooming test after modeling. Immunofluorescence and hematoxylin and eosin staining were utilized to determine the content of reactive oxygen species, activation of microglial cells, neuroprotection, and nerve regeneration. Compared with the control group, the L-Theanine + NEP1-40 group had significantly decreased myeloperoxidase, malondialdehyde, interleukin-6, reactive oxygen species, and microglial cells, significantly increased score on the Terzis grooming test, increased motor neuron content, and thickened muscle fibers, increased area, and appearance of large and clear motor endplate structures. The results of this study suggest that l-Theanine combined with NEP1-40significantly promoted nerve regeneration after brachial plexus root avulsion, and may be a potential treatment for promoting nerve regeneration. Possible mechanisms underlying these results are alleviation of oxidative damage and inflammatory responses in the injured area and antagonism of myelin inhibition.