Major depressive disorder is typically treated with selective serotonin reuptake inhibitors (SSRIs), however, SSRIs take approximately six weeks to produce therapeutic effects, if any. Not surprisingly, there has been great interest in findings that low doses of ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, produce rapid and long-lasting antidepressant effects. Preclinical studies show that the antidepressant-like effects of ketamine are dependent upon availability of serotonin, and that ketamine increases extracellular serotonin, yet the mechanism by which this occurs is unknown. Here we examined the role of the high-affinity, low-capacity serotonin transporter (SERT), and the plasma membrane monoamine transporter (PMAT), a low-affinity, high-capacity transporter for serotonin, as mechanisms contributing to ketamine’s ability to increase extracellular serotonin and produce antidepressant-like effects. Using high-speed chronoamperometry to measure real-time clearance of serotonin from CA3 region of hippocampus in vivo, we found ketamine robustly inhibited serotonin clearance in wild-type mice, an effect that was lost in mice constitutively lacking SERT or PMAT. As expected, in wild-type mice, ketamine produced antidepressant-like effects in the forced swim test. Mapping onto our neurochemical findings, the antidepressant-like effects of ketamine were lost in mice lacking SERT or PMAT. Future research is needed to understand how constitutive loss of either SERT or PMAT, and compensation that occurs in other systems, is sufficient to void ketamine of its ability to inhibit serotonin clearance and produce antidepressant-like effects. Taken together with existing literature, a critical role for serotonin, and its inhibition of uptake via SERT and PMAT, cannot be ruled out as important contributing factors to ketamine’s antidepressant mechanism of action. Combined with what is already known about ketamine’s action at NMDA receptors, these studies help lead the way to the development of drugs that lack ketamine’s abuse potential but have superior efficacy in treating depression.

Background Efficient DNA repair in response to standard chemo and radiation therapies often contribute to GBM therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double strand break (DSB) repair and combination of KDM1A inhibitor and TMZ in vitro and in vivo using patient derived GSCs. Methods Brain-bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. Effect of combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, Western blot, HR and NHEJ reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. Results TCGA analysis showed KDM1A is highly expressed in TMZ treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and selfrenewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the BBB. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ mediated DNA damage. Combination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced survival of tumor bearing mice. Conclusions Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

The poor effectiveness of antidepressants is hypothesized to be attributable, in part, to high volume transporters with low selectivity (i.e., “uptake‐2” mechanisms) that undermine antidepressant blockade of highly selective, low volume transporters (i.e., “uptake‐1” transporters) such as the serotonin transporter. Compared to other uptake‐2 transporters in brain, plasma membrane monoamine transporter (PMAT, Slc29a4) preferentially transports serotonin and dopamine, both heavily implicated in the pathophysiology of depression. Therefore, we hypothesized that reduced function of PMAT would enhance the ability of antidepressants to elicit antidepressant‐like behaviors in a forced swim or tail suspension test, and to impair clearance of extracellular serotonin. Because a selective pharmacologic inhibitor of PMAT has yet to be identified, genetic knockout of PMAT is currently the best available method for investigating PMAT's functional role. Using a mouse line recently developed in the lab of Dr. Joanne Wang, we compared male and female wildtype (+/+) controls against mice with reduced (+/−) or completely ablated (−/−) PMAT function to evaluate how PMAT deficiency affects behavioral responses to antidepressants in the forced swim and tail suspension tests. Sub‐effective doses of the serotonin transporter inhibitors fluvoxamine or escitalopram, or the dopamine/norepinephrine transporter inhibitor bupropion, were given 30 min prior to a forced swim or tail suspension test. Behaviors were scored by observers blind to sex, genotype, and treatment. Preliminary findings indicate that male −/− mice may selectively exhibit antidepressant‐like responses to bupropion and fluvoxamine through an increase in swimming behavior. In contrast, female −/− mice appear to exhibit a depressive‐like response specifically to bupropion. These studies are continuing. Ongoing experiments are measuring serotonin clearance in the nucleus accumbens of male +/+ and −/− mice using in vivo high‐speed chronoamperometry in the presence or absence of uptake‐1 inhibitors. These initial results support our hypothesis and suggest an unexpected sex‐specific contribution of PMAT function in the poor effectiveness of uptake‐1 targeting antidepressant drugs. Though chronoamperometry experiments are still in early stages, these will afford insight into possible neurochemical differences that could explain these intriguing sex differences. Given our early behavioral findings, greater focus on drug discovery for PMAT‐selective inhibitors could reveal compounds that are useful as antidepressant adjuvants. Future work will focus on identifying potential mechanisms through which these sex‐ and genotype‐dependent antidepressant responses are mediated.Support or Funding InformationThis work was supported by a Brain & Behavior Research Foundation and Vital Projects Fund, Inc., NARSAD Young Investigator Grant (26249) to TLG and National Institute of Mental Health grants (R01 MH093320 and R01 MH106978) to LCD. TLG is supported by a National Institute on Drug Abuse grant (T32 DA031115) to Charles P. France.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background and PurposeTau pathology contributes to a bidirectional relationship between sleep disruption and neurodegenerative disease. Tau transgenic rTg4510 mice model tauopathy symptoms including sleep/wake disturbances, which manifest as marked hyperarousal. This phenotype can be prevented by early transgene suppression, however, whether hyperarousal can be rescued after its onset is unknown.Experimental ApproachThree chronic (8‐week) experiments were conducted with wild‐type and rTg4510 mice after the age of onset of hyperarousal (4.5 months): 1) Tau transgene suppression with doxycycline (200 ppm); 2) Inactive phase REM sleep enhancement with the dual orexin receptor antagonist suvorexant (50 mg/kg/day); or 3) Active phase NREM and REM sleep enhancement using the selective orexin 2 (OX2) receptor antagonist MK‐1064 (40 mg/kg/day). Sleep was assessed using polysomnography, cognition using the Barnes maze, and tau pathology using western blot and/or immunohistochemistry.Key ResultsTau transgene suppression improved tauopathy and hippocampal‐dependent spatial memory, but didn’t modify hyperarousal. Pharmacological rescue of REM sleep deficits did not improve spatial memory or tau pathology. In contrast, normalising hyperarousal by increasing both NREM and REM sleep via OX2 receptor antagonism restored spatial memory, independently of tauopathy, but only in male rTg4510 mice. OX2 receptor antagonism induced only short‐lived hypnotic responses in female rTg4510 mice and did not improve spatial memory, indicating a tau‐ and sex‐dependent disruption of OX2 receptor signalling.Conclusions and ImplicationsPharmacologically reducing hyperarousal corrects tau‐induced sleep/wake and cognitive deficits. However, tauopathy causes sex‐dependent disruptions of OX2 receptor signalling/function, which may have implications for choice of hypnotic therapeutics in tauopathies.