Timothy J. Maher’s research while affiliated with Boston University and other places

Environmental and occupational exposure to heavy metals remains one of the major concerns in public health. Increased levels of manganese (Mn) pollution are associated with profound neurotoxic effects, including neurobehavioral deficits and disturbances resembling Parkinson’s disease. While Mn absorption is in part mediated by iron transporters, recent studies have shown that the levels of iron transporters are modified by alcohol and that chronic alcohol consumption increases body iron stores. However, it is largely unexplored whether alcohol exposure influences the transport and neurotoxicity of Mn. To address this question, we exposed mice to ethanol (10%; v/v) by drinking water for 4 weeks, during which period MnCl2 (5 mg/kg) or saline solutions were administered daily by intranasal instillation. Ethanol consumption in mice increased brain Mn levels in a dose-dependent manner after Mn instillation, determined by inductively-coupled plasma mass spectrometry, which was accompanied by up-regulation of iron transporters, as assessed by western blotting and qPCR. In addition, alcohol drinking increased hypoxic response and decreased hepcidin expression, providing the molecular mechanism of increased iron transporters and Mn uptake upon alcohol consumption. Moreover, brain dopamine levels, analyzed by HPLC, were decreased after intranasal Mn instillation, which was worsened by alcohol. Likewise, alcohol-Mn co-exposure synergistically altered dopaminergic protein expression. Finally, alcohol binge-drinking, which resembles alcohol drinking manner in humans, increased brain Mn content along with upregulation of iron transporters. Our study suggests that individuals who consume alcohol may have a higher risk of Mn neurotoxicity upon Mn exposure.

In a search for new neuropeptide S receptor antagonists, we have described a new series of quinolone-pyranopyrimidine hybrid derivatives aiming to modify the inhibitory characters towards NPSR to develop new therapeutic strategies against anxiety, addiction and food disorders. We identified six potent antagonists 3, 4b, 6, 8, 9 and 10 which counteracted the stimulatory effect of NPS at both Gq and Gs pathways, at low micromolar concentrations, through modulation of Ca²⁺ and cAMP signaling, respectively. Molecular docking predicted the orientation mode of the top active compounds; 10 and 4b with ΔG value of -23.94 and -23.87 kcal/mol, respectively that is considered good when compared to that of the reference compound ML154 (ΔG =-25.75) kcal/mol. Molecular dynamic simulations confirmed the stability of binding of compound 10 to the homology model of NPSR as it reached the equilibrium after 4 ns at RMSD of 1.00 Å while ML154 was faster to achieve the equilibrium after 2 ns at RMSD of 1.00 Å.

Triple negative breast cancer (TNBC) is one of the most aggressive types of breast cancer accounting for 12% of breast cancer cases. It is characterized by the lack of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER‐2), which limits treatment options and enhances its ability to metastasize with the risk of recurrence. Patients with TNBC are not responsive to conventional therapies. Capsaicin (CAP) is the most abundant capsaicinoid produced in chili pepper fruits and has been utilized for its analgesic and anti‐inflammatory effects. While several studies have demonstrated that capsaicin has anti‐carcinogenic properties in various types of human cancers, the underlying molecular mechanisms remain to be explored. The aim of this study was to investigate the effects of capsaicin in human TNBC, utilizing a BT‐20 cell model. Capsaicin demonstrated concentration‐ and time‐dependent in the viability of BT‐20 cells, as determined by the MTS assay. Capsaicin caused significant increases in cytochrome C release, caspase 3/7 activity and expression of cleaved poly‐(ADP‐ribose) polymerase (PARP), all of which are markers of apoptotic activation. These effects were accompanied by down‐regulation of cyclin D1 production, an indicator of cell cycle arrest at the G0/G1 phase. Further analyses of signaling mechanisms revealed that capsaicin significantly inhibited EGFR phosphorylation and the phosphorylation of its downstream signaling proteins AKT and MAPK, which may provide some explanation for its mechanism of action in TNBC. In conclusion, capsaicin has demonstrated inhibitory effects on cell growth and cell cycle progression on TNBC cells by enhancing apoptosis. Our data demonstrated a novel mechanism for capsaicin, modulating EGFR signaling by AKT/MAPK pathways in BT‐20 cells. These results provide useful information relevant to the development of a potential new therapy to treat TNBC with capsaicin. Support or Funding Information This work was funded by the College of Medicine, King Saud bin Abdulaziz University for Health Sciences (KSAU‐HS), Jeddah, Saudi Arabia. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The pentane extract of the Peruvian plant, Lepidium meyenii (Maca), has been demonstrated to possess neuroprotective activity in previous in vitro and in vivo studies (Pino-Figueroa et al. in Ann N Y Acad Sci 1199:77–85, 2010; Pino-Figueroa et al. in Am J Neuroprot Neuroregener 3:87–92, 2011). This extract contains a number of macamides that may act on the endocannabinoid system (Pino-Figueroa et al. in Ann N Y Acad Sci 1199:77–85, 2010; Pino-Figueroa et al., 2011; Dini et al. in Food Chem 49:347–349, 1994). The aim of this study was to characterize the inhibitory activity of four of these maccamides (N-benzylstearamide, N-benzyloleamide, N-benzyloctadeca-9Z,12Z-dienamide, and N-benzyloctadeca-9Z,12Z,15Z-trienamide) on fatty acid amide hydrolase (FAAH), an enzyme that is responsible for endocannabinoid degradation in the nervous system (Kumar et al. in Anaesthesia 56:1059–1068, 2001). The four compounds were tested at concentrations between 1 and 100 μM, utilizing an FAAH inhibitor screening assay. The results demonstrated concentration-dependent FAAH inhibitory activities for the four macamides tested. N-Benzyloctadeca-9Z,12Z-dienamide demonstrated the highest FAAH inhibitory activity whereas N-benzylstearamide had the lowest inhibitory activity. In addition, N-benzylstearamide, N-benzyloleamide, and N-benzyloctadeca-9Z,12Z-dienamide demonstrated time-dependent inhibition when tested after a pre-incubation period, indicating that the mechanism of inhibition for these compounds most likely is irreversible. Of interest, unsaturation in the fatty acid moiety resulted in greater FAAH inhibitory activity. LC/MS/MS analysis demonstrated that FAAH was able to hydrolyze N-benzyloctadeca-9Z,12Z-dienamide, suggesting that N-benzyloctadeca-9Z,12Z-dienamide is also a slow substrate for FAAH. These results provide useful information about the mechanism of action of Lepidium meyenii and may help with the development of new compounds with FAAH inhibitory or modulatory activity.

Ubiquitin proteasome system (UPS) is a conserved proteolytic process involved in the degradation of damaged and misfolded proteins. Proteins undergoing this event must be conjugated to one or more monomers of ubiquitin, a process known as ubiquitination. UPS inhibition produces protein aggregation which causes cytotoxicity. Low‐density lipoprotein receptor‐related protein‐1 (LRP1) is a transmembrane receptor composed of two subunits, α (515 kDa) and β (85 kDa) located at extracellular and intracellular levels, respectively. This receptor is involved in the uptake of molecules via clathrin for either transcellular transport or lysosomal degradation. UPS inhibition by MG132 increases LRP1 half‐life in HepG2 cells as reported in the literature. Conversely, protein aggregation might have a role in LRP1 reduction. The purpose of this research was to demonstrate that MG132, a UPS inhibitor, induces cytotoxicity, protein aggregation and reduces LRP1 protein levels in HepG2 cells. These cells were treated for 24 hours with different concentrations of MG132 (0.03 – 3.0 μM) reducing cell viability in a concentration‐dependent manner determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS assay). Whole cell lysates were extracted and separated on SDS‐PAGE. Immunoblotting was used to determine K48‐linked ubiquitinated proteins and LRP1. MG132 at 0.3 and 1 μM increased K48‐linked ubiquitinated proteins and reduced LRP1 protein levels in a concentration‐dependent manner. These results suggest that UPS inhibition promotes protein aggregation, which can be the cause of cytotoxicity. In addition, MG132 reduced LRP1 protein levels which might be a consequence of protein aggregate accumulation. Further experiments are required to elucidate the LRP1 reduction mechanism when UPS is inhibited.

LRP1 is a transmembrane receptor involved in the internalization of molecules by either transcellular transport or lysosomal degradation via clathrin. PPARγ is a nuclear receptor that transcribes several target genes involved in cell differentiation, lipid metabolism and glucose homeostasis. PPARγ activation has been shown to upregulate LRP1 mRNA and protein levels in HepG2 cells. However, overactivation of this nuclear receptor has exhibited a reduction in LRP1 protein levels. The aim of this research was to evaluate the effect of rosiglitazone (RGZ), a known PPARγ agonist, on LRP1 protein levels in HepG2 cells. Additionally, the LRP1 protein reduction mechanism in RGZ treated‐cells studying PPARγ activation, ubiquitin proteasome system and lysosomal degradation were evaluated. HepG2 cells were treated with RGZ (0.003 – 30 μM) to show no increase on LRP1 protein levels as reported in the literature. However, cells incubated for 24 and 48 hours with 30 μM of RGZ reduced LRP1 protein levels by 40 and 90%, respectively. PPARγ antagonism and ubiquitin proteasome system inhibition were studied in RGZ treated‐cells demonstrating no effect on LRP1 reduction. Bafilomycin A1, a lysosome degradation inhibitor, increased LRP1 protein levels up to 4‐fold in RGZ treated‐cells. These results suggest that overactivation of PPARγ reduces LRP1 protein levels via lysosome degradation. Further experiments are required to evaluate the mechanism involved in the effect of RGZ on LRP1 protein expression.

Iron deficiency is closely associated with altered GABA metabolism and affective behavior. While mutation in the hemochromatosis (HFE) gene disrupts iron homeostasis and promotes oxidative stress that increases the risk of neurodegeneration, it is largely unknown whether HFE mutation modifies GABAergic homeostasis and emotional behavior. The goal of our study was to investigate the impact of HFE on GABAergic neurochemistry and redox–epigenetic regulation in the brain using H67D HFE‐mutant mice that recapitulates the H63D‐HFE mutation in humans. H67D mice displayed elevated redox‐active iron levels in the brain by 32% compared to age‐matched wild‐type mice. Moreover, the H67D brain had increased isoprostane and decreased glutathione, indicating elevated oxidative stress. Additionally, the H67D brain had decreased global methylation and attenuated DNA methyltransferase (DNMT) activity. Direct addition of iron to purified DNMT in vitro decreased enzyme activity in a concentration‐dependent manner. Last, H67D mice exhibited decreased anxiety‐like behavior, which was associated with increased expression of the GABAA receptor α2 subunits by 93%, and these changes were also observed in H67D mice fed a low‐iron diet. Taken together, our results suggest a putative role of HFE in regulating labile iron status in the brain, and mutation in H67D perturbs redox‐methylation status, contributing to GABAergic dysfunction.—Ye, Q., Trivedi, M., Zhang, Y., Böhlke, M., Alsulimani, H., Chang, J., Maher, T., Deth, R., Kim, J. Brain iron loading impairs DNA methylation and alters GABAergic function in mice. FASEB J. 33, 2460–2471 (2019). www.fasebj.org

Purpose Pentane extract of the Peruvian plant Lepidium meyenii (Maca) has been demonstrated to possess neuroprotective activity in previous studies. This extract contains several macamides that might act on the endocannabinoid system. The aim of this study is to characterize the inhibitory activity of four maccamides ( N ‐benzylstearamide, N ‐benzyloleamide, N‐ benzyloctadeca‐9 Z ,12 Z ‐dienamide, and N ‐benzyloctadeca‐9 Z ,12 Z ,15 Z ‐trienamide) on fatty acid amide hydrolase (FAAH), an enzyme that is responsible for the endocannabinoid degradation in the nervous system. The enhancement of endocannabinoid activity by inhibition of FAAH enzyme results in analgesic, anxiolytic, anti‐depressant and neuroprotectant effects, avoiding unwanted side effects of cannabinoid receptor activation such as weight gain and motor cognitive impairments. Methods The four compounds at concentrations from 1 to 100 μM were tested using a FAAH inhibitor screening assay method. This assay is based on fluorescence produced upon the hydrolysis of a substrate, 7‐amino‐4‐methylcoumarin‐arachidonamide (AMC‐AA) by the FAAH enzyme to the 7‐amino‐4‐methylcoumarin (AMC) which is measured 60 min following substrate addition at 37ºC with excitation and emission of 360 and 460 nm, respectively. To study the time dependence effect on the FAAH enzyme inhibition, the previous assay was repeated using different pre‐incubation times (0, 60 and 120 min) before substrate addition at 37ºC. To evaluate whether macamides are purely inhibitors or might be substrates of FAAH, LC/MS/MS was used to analyze the 60 min incubation of N ‐benzyloctadeca‐9 Z ,12 Z ‐dienamide with and without FAAH enzyme at 37ºC. The benzylamine was used as a standard to determine the possible degradation of N ‐benzyloctadeca‐9 Z ,12 Z ‐dienamide by FAAH enzyme. Results The four macamides studied demonstrated concentration‐dependent FAAH inhibitory activity. Double bonds appeared to have an important impact on FAAH inhibitory activity. The macamides containing oleic, linoleic and linolenic acids produced FAAH inhibition of 64, 73, and 54%, respectively, while the macamide containing stearic acid and lacking a double bond produced only 13% inhibition at 100 μM. N ‐Benzyloctadeca‐9Z,12Z‐dienamide (two double bonds) demonstrated the greatest inhibitory activity, indicating that two double bonds significantly influence the inhibition of FAAH. Pre‐incubation studies showed significant differences in the FAAH inhibitory activity of N ‐benzyloctadeca‐9Z,12Z‐dienamide, N‐ benzyloleamide, and N ‐benzylstearamide, indicating that the inhibitory mechanism of these compounds is time‐dependent. Incubation of N ‐benzyloctadeca‐9Z,12Z‐dienamide with FAAH enzyme for 60 min resulted in a 44% degradation, when analyzed by LC/MS/MS. This result indicates that N ‐benzyloctadeca‐9Z,12Z‐dienamide likely acts as a slow substrate for the FAAH enzyme. Conclusion N ‐Benzyloctadeca‐9 Z ,12 Z ‐dienamide is the most active macamide. The presence of double bonds in macamides significantly increases FAAH inhibition, which could improve the potential neuroprotective and analgesic activities. These results will provide useful information regarding the development of new compounds with FAAH inhibitory activity. Support or Funding Information King Saud ben Abdulaziz University for Health Sciences (KSAU‐HS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Macamides are a distinct class of secondary metabolites, benzylamides of long chain fatty acids, which were isolated from the Peruvian plant Lepidium meyenii (Maca). As structural analogues of the endocannabinoid anandamide (AEA), they have demonstrated neuroprotective effects in vitro and in vivo. The purpose of this study was to demonstrate the neuroprotective activity of the macamides: N-(3-methoxybenzyl)oleamide (MAC 18:1), N-(3-methoxybenzyl)linoleamide (MAC 18:2) and N-(3-methoxybenzyl)linolenamide (MAC 18:3) in a neurotoxic environment caused by exposure of U-87 MG glioblastoma cells to manganese chloride (MnCl2). The neuroprotective effects of these macamides were reversed by the CB1 antagonist AM251. The mechanism by which manganese (Mn) induces cell damage was investigated by studying its effects on mitochondria. Reactive oxygen species (ROS) increase intracellular calcium and enhance the opening of mitochondrial permeability transition pores (MPTP), which leads to decreased mitochondrial membrane potential (MMP), to disruption of mitochondria and to neuron death in neurodegenerative disorders. In this study, MnCl2 at 50μM was responsible for mitochondrial disruption, which was attenuated by all three of the macamides tested. Human peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to be a cannabinoid target, and PPARγ has also been demonstrated to mediate some of the longer-term vascular effects of the plant cannabinoid, ∆9-tetrahydrocannabinol. PPARγ activation was observed in response to exposures of cells to MAC 18:2 and MAC 18:3. These findings suggest that macamides achieve their neuroprotective effects by binding to CB1 receptors to protect against Mn-induced toxicity in U-87 MG glioblastoma cells. Additionally these macamides, in a manner similar to the analogous endocannabinoid AEA, interact with other targets such as PPARγ to regulate metabolism and energy homeostasis, cell differentiation and inflammation.

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor involved in the uptake of a variety of molecules, such as apoE, α2-macroglobulin, and the amyloid β peptide (Aβ), for either transcellular transport, protein trafficking or lysosomal degradation. The LRP1 gene can be transcribed upon activation of peroxisome proliferator receptor activated-γ (PPARγ) by the potent PPARγ agonist, rosiglitazone (RGZ). In previous studies, RGZ was shown to upregulate LRP1 levels in concentrations between 0.1 and 5 μM in HepG2 cells. In this study, we sought to replicate previous studies and to investigate the molecular mechanism by which high concentrations of RGZ reduce LRP1 levels in HepG2 cells. Our data confirmed that transcriptional activation of LRP1 occurred in response to RGZ at 3 and 10 μM, in agreement with the study reported by Moon and collaborators. On the other hand, we found that high concentrations of RGZ decreased both mRNA and protein levels of LRP1. Mechanistically, transcriptional dysregulation of LRP1 was affected by the downregulation of PPARγ in a time- and concentration-dependent manner. However, downregulation of PPARγ was responsible for only 40% of the LRP1 reduction and thereby the remaining loss of LRP1 (60%) was found to be through degradation in the lysosomal system. In conclusion, our findings demonstrate the mechanisms by which high concentrations of RGZ caused LRP1 levels to be reduced in HepG2 cells. Taken together, this data will be helpful to better explain the pharmacological modulation of this pivotal membrane receptor by PPARγ agonists. Keywords: LRP1, rosiglitazone, PPARγ, protein degradation, lysosomal degradation, Bafilomycin A1.