A Human TREK-1/HEK Cell Line: A Highly Efficient Screening Tool for Drug Development in Neurological Diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model

Regulation of serum spadin propeptide: An antidepressant response probe

ObjectivesWe previously discovered that spadin, a short analogue of the propeptide (PE) released from the maturation of sortilin, displays potent antidepressant properties. Since the PE level can be measured in the blood, we aimed to investigate how the PE serum concentration is regulated in mice. We wondered whether the PE serum levels vary between healthy subjects and patients with major depressive disorder (MDD).MethodsWe developped a dosing method based on the AlphaScreen™ technology (Perkin) which allow to selectively detect both PE, spadin and metabolic products from these peptides with a detection range of 1 ng/mL.ResultsWe found that insulin significantly up-regulated serum PE concentration from 26.15 ± 2.63 to 41.43 ± 6.27 nM (P = 0.0318). Analysis during circadian cycle in mice revealed that the amount of PE and its derivatives significantly varied during the cycle being higher during the period of maximal activity (dark period). We also measured serum insulin concentration between 1 and 7 pm and observed a significant rise confirming the relationships between insulin and PE concentration. We showed that the serum level of PE is lower in depressive patients than in healthy non-psychiatric. We observed that the weaker level of PE in depressive patients can recover the level of healthy subjects after a chronic antidepressant treatment.ConclusionsDosing the serum level of PE could be a promising approach for the diagnosis of depression and to determine the remission of the disease.Disclosure of interestThe authors have not supplied their declaration of competing interest.</jats:sec

The Mini-Spadin, an efficient alternate to Spadin in the depression treatment

ObjectivesWe previously discovered spadin as a new antidepressant drug concept. Spadin exerts its antidepressant actions on the TREK-1 potassium channel, a new antidepressant (AD) target. We have shown that spadin acts more rapidly in comparison to other ADs. We have pointed out that spadin induced neurogenesis after only 4-day treatments. We have demonstrated that spadin did not display side effects at the cardiac level and on TREK-1 controlled functions such as stroke, epilepsy or pain.ObjectivesWith the final goal to make spadin a drug for human clinic, our objective was to find analogs of spadin demonstrating a better affinity or a better in vivo stability or both.MethodsSeveral analogs of spadin were synthesized. Their ability to block the TREK-1 channel activity were first tested by electrophysiology on HEK293 cells stably transfected with TREK-1 channels. AD effects were measured by using the forced swim test and the novelty suppressed feeding test. Neurogenesis was investigated by measuring the expression level of the synaptic protein PSD-95 in in vitro cultured neurons.ResultsOur data allow us to identify a shortened spadin, called mini-spadin, that displayed the same AD properties as spadin and a 400 fold increase in the TREK-1 affinity. Mini-spadin increased the synaptogenesis marker PSD95 levels after only 24 hours of treatment, suggesting that like spadin, mini-spadin was able to induce neurogenesis and synaptogenesis.ConclusionsEven if further experiments are required, the mini-spadin appears to be more efficient than spadin offering a very promising alternate to spadin as human drug.Disclosure of interestThe authors have not supplied their declaration of competing interest.</jats:sec

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs.

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components

NCCN Chemotherapy Order Templates: Enhancing patient safety through standardization

e17537 Background: NCCN Chemotherapy Order Templates (COT), which delineate antineoplastics and associated supportive care agents, monitoring and safety parameters, and instructions for self-administered agents, were launched in 2008 to complement the NCCN Clinical Practice Guidelines in Oncology and NCCN Drugs and Biologics Compendium for nine cancers. Through COT, NCCN sought to enhance patient safety by reducing medication errors, anticipating and managing adverse events, and standardizing care. NCCN developed and distributed a survey to evaluate the reception, use, and impact of COT on oncology practice. Methods: The survey included multiple-choice questions with filtering logic. Questions included 5-point Likert scale. The survey was emailed on December 18, 2008 to a convenience sample of 10,183 registered users of NCCN.org who had clicked at least once to the COT; 110 (1%) survey emails were undeliverable. 734 (7%) responded by 1/5/09 and 588 (6%) completed the survey (80% completion rate). 28 (&lt;1%) recipients opted out of participating. Results: 476 (64%) respondents were providers, including MDs (47%), mid-level (8%) and nurses (10%); 11% were pharmacists. Of the 734 respondents, 465 (63%) had used COT. Among the 465 users, the median number COT accessed was 2 (range 0 - 9) with physicians accessing more than other providers (p = 0.002). Breast cancer COT were accessed most (68%). COT were used as a reference (52%) and for setting up chemotherapy orders (42%). 292 users (63%) agreed that COT impacted patient safety; with MDs more likely to agree (p = 0.001) compared to other providers. Of these, 89% responded that COT made chemotherapy ordering safer. 271 users (58%) agreed that using NCCN COT in practice impacted the ordering of supportive care agents. Among providers, MDs were more likely to agree (p = 0.002). Of those who agreed, 86% responded that COT made it more likely that appropriate supportive care orders were included. Conclusions: Adding NCCN Chemotherapy Order Templates to the core NCCN content was done to make the NCCN Clinical Practice Guidelines in Oncology more accessible to providers. Survey results indicate that COT users feel that template use improves the safety of chemotherapy and supportive care agents. Throughout 2009, NCCN will continue to develop COT to cover most cancers. [Table: see text] </jats:p

Astroglial Control of the Antidepressant-Like Effects of Prefrontal Cortex Deep Brain Stimulation

AbstractAlthough deep brain stimulation (DBS) shows promising efficacy as a therapy for intractable depression, the neurobiological bases underlying its therapeutic action remain largely unknown. The present study was aimed at characterizing the effects of infralimbic prefrontal cortex (IL-PFC) DBS on several pre-clinical markers of the antidepressant-like response and at investigating putative non-neuronal mechanism underlying DBS action. We found that DBS induced an antidepressant-like response that was prevented by IL-PFC neuronal lesion and by adenosine A1 receptor antagonists including caffeine. Moreover, high frequency DBS induced a rapid increase of hippocampal mitosis and reversed the effects of stress on hippocampal synaptic metaplasticity. In addition, DBS increased spontaneous IL-PFC low-frequency oscillations and both raphe 5-HT firing activity and synaptogenesis. Unambiguously, a local glial lesion counteracted all these neurobiological effects of DBS. Further in vivo electrophysiological results revealed that this astrocytic modulation of DBS involved adenosine A1 receptors and K+ buffering system. Finally, a glial lesion within the site of stimulation failed to counteract the beneficial effects of low frequency (30Hz) DBS. It is proposed that an unaltered neuronal–glial system constitutes a major prerequisite to optimize antidepressant DBS efficacy. It is also suggested that decreasing frequency could heighten antidepressant response of partial responders