Circadian rhythms mediate malaria transmission potential.

Malaria transmission begins when infected female Anopheles mosquitos deposit Plasmodium parasites into the mammalian host's skin during a bloodmeal. The salivary gland-resident sporozoite parasites migrate to the bloodstream, subsequently invading and replicating within hepatocytes. As Anopheles mosquitos are more active at night, with a 24-hour rhythm, we investigated whether their salivary glands are under circadian control, anticipating bloodmeals and modulating sporozoite biology for host encounters. Here we show that approximately half of the mosquito salivary gland transcriptome, particularly genes essential for efficient bloodmeals such as anti-blood clotting factors, exhibits circadian rhythmic expression. Furthermore, we demonstrate that mosquitoes prefer to feed during nighttime, with the amount of blood ingested varying cyclically throughout the day. Notably, we show a substantial subset of the sporozoite transcriptome cycling throughout the day. These include genes involved in parasite motility, potentially modulating the ability to initiate infection at different times of day. Thus, although sporozoites are typically considered quiescent, our results demonstrate their transcriptional activity, revealing robust daily rhythms of gene expression. Our findings suggest a circadian evolutionary relationship between the vector, parasite and mammalian host that together modulate malaria transmission.

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

The Molecular Mechanism of Positive Allosteric Modulation at the Dopamine D1 Receptor.

The dopamine D1 receptor (D1R) is a promising target for treating various psychiatric disorders. While upregulation of D1R activity has shown potential in alleviating motor and cognitive symptoms, orthosteric agonists have limitations, restricting their clinical applications. However, the discovery of several allosteric compounds specifically targeting the D1R, such as LY3154207, has opened new therapeutic avenues. Based on the cryo-EM structures of the D1R, we conducted molecular dynamics simulations to investigate the binding and allosteric mechanisms of LY3154207. Our simulations revealed that LY3154207 preferred the horizontal orientation above intracellular loop 2 (IL2) and stabilized the helical conformation of IL2. Moreover, LY3154207 binding induced subtle yet significant changes in key structural motifs and their neighboring residues. Notably, a cluster of residues centered around the Na+-binding site became more compact, while interactions involving the PIF motif and its neighboring residues were loosened upon LY3154207 binding, consistent with their role in opening the intracellular crevice for receptor activation. Additionally, we identified an allosteric pathway likely responsible for the positive allosteric effect of LY3154207 in enhancing Gs protein coupling. This mechanistic understanding of LY3154207's allosteric action at the D1R paves the way for the rational design of more potent and effective allosteric modulators.

The molecular mechanism of positive allosteric modulation at the dopamine D1 receptor.

The dopamine D1 receptor (D1R) is a promising target for treating various psychiatric disorders. While upregulation of D1R activity has shown potential in alleviating motor and cognitive symptoms, orthosteric agonists have limitations, restricting their clinical applications. However, the discovery of several allosteric compounds specifically targeting the D1R, such as LY3154207, has opened new therapeutic avenues. Based on the cryo-EM structures of the D1R, we conducted molecular dynamics simulations to investigate the binding and allosteric mechanisms of LY3154207. Our simulations revealed that LY3154207 preferred the horizontal orientation above intracellular loop 2 (IL2) and stabilized the helical conformation of IL2. Moreover, LY3154207 binding induced subtle yet significant changes in key structural motifs and their neighboring residues. Notably, a cluster of residues centered around the Na + binding site became more compact, while interactions involving the PIF motif and its neighboring residues were loosened upon LY3154207 binding, consistent with their role in opening the intracellular crevice for receptor activation. Additionally, we identified an allosteric pathway likely responsible for the positive allosteric effect of LY3154207 in enhancing Gs protein coupling. This mechanistic understanding of LY3154207's allosteric action at the D1R pave the way for the rational design of more potent and effective allosteric modulators.

Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic and targetable tissue states.

Glioblastoma (GBM) diffusely infiltrates the brain and intermingles with non-neoplastic brain cells, including astrocytes, neurons and microglia/myeloid cells. This complex mixture of cell types forms the biological context for therapeutic response and tumor recurrence. We used single-nucleus RNA sequencing and spatial transcriptomics to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional 'tissue-states' defined by cohabitation patterns between specific subpopulations of neoplastic and non-neoplastic brain cells. These tissue-states correlated with radiographic, histopathologic, and prognostic features and were enriched in distinct metabolic pathways. Fatty acid biosynthesis was enriched in the tissue-state defined by the cohabitation of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and macrophages, and was associated with recurrent GBM and shorter survival. Treating acute slices of GBM with a fatty acid synthesis inhibitor depleted the transcriptional signature of this pernicious tissue-state. These findings point to therapies that target interdependencies in the GBM microenvironment.

Binding preference at the µ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl.

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining µ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated ß-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

A Journey through Diastereomeric Space: The Design, Synthesis, In Vitro and In Vivo Pharmacological Activity, and Molecular Modeling of Novel Potent Diastereomeric MOR Agonists and Antagonists.

Four sets of diastereomeric C9-alkenyl 5-phenylmorphans, varying in the length of the C9-alkenyl chain, were designed to examine the effect of these spatially distinct ligands on opioid receptors. Functional activity was obtained by forskolin-induced cAMP accumulation assays and several compounds were examined in the [35S]GTPgS assay and in an assay for respiratory depression. In each of the four sets, similarities and differences were observed dependent on the length of their C9-alkenyl chain and, most importantly, their stereochemistry. Three MOR antagonists were found to be as or more potent than naltrexone and, unlike naltrexone, none had MOR, KOR, or DOR agonist activity. Several potent MOR full agonists were obtained, and, of particular interest partial agonists were found that exhibited less respiratory depression than that caused by morphine. The effect of stereochemistry and the length of the C9-alkenyl chain was also explored using molecular modeling. The MOR antagonists were found to interact with the inactive (4DKL) MOR crystal structures and agonists were found to interact with the active (6DDF) MOR crystal structures. The comparison of their binding modes at the mouse MOR was used to gain insight into the structural basis for their stereochemically induced pharmacological differences.

De Novo Design of Molecules with Low Hole Reorganization Energy Based on a Quarter-Million Molecule DFT Screen: Part 2.

Organic semiconductors have many desirable properties including improved manufacturing and flexible mechanical properties. Due to the vastness of chemical space, it is essential to efficiently explore chemical space when designing new materials, including through the use of generative techniques. New generative machine learning methods for molecular design continue to be published in the literature at a significant rate but successfully adapting methods to new chemistry and problem domains remains difficult. These challenges necessitate continual method evaluation to probe method viability for use in alternative applications not covered in the original works. In continuation of our previous work, we evaluate four additional machine-learning-based de novo methods for generating molecules with high predicted hole mobility for use in semiconductor applications. The four generative methods evaluated here are (1) Molecule Deep Q-Networks (MolDQN), which utilizes Deep-Q learning to directly optimize molecular structure graphs for desired properties instead of generating SMILES, (2) Graph-based Genetic Algorithm (GraphGA), which uses a genetic algorithm for optimization where crossovers and mutations are defined in terms of RDKit's reaction SMILES, (3) Generative Tensorial Reinforcement Learning (GENTRL), which is a variational autoencoder (VAE) with a learned prior distribution and optimized using reinforcement learning, and (4) Monte Carlo tree search exploration of chemical space in conjunction with a recurrent neural network (RNN) decoder (ChemTS). The generated molecules were evaluated using density functional theory (DFT) and we discovered better performing molecules with the GraphGA method compared to the other approaches.

Single unit analysis and wide-field imaging reveal alterations in excitatory and inhibitory neurons in glioma.

While several studies have attributed the development of tumour-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neuron in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumour-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation.

A comprehensive evaluation of the potential binding poses of fentanyl and its analogs at the µ-opioid receptor.

Fentanyl and its analogs are selective agonists of the µ-opioid receptor (MOR). Among novel synthetic opioids (NSOs), they dominate the recreational drug market and are the main culprits for the opioid crisis, which has been exacerbated by the COVID-19 pandemic. By taking advantage of the crystal structures of the MOR, several groups have investigated the binding mechanism of fentanyl, but have not reached a consensus, in terms of both the binding orientation and the fentanyl conformation. Thus, the binding mechanism of fentanyl at the MOR remains an unsolved and challenging question. Here, we carried out a systematic computational study to investigate the preferred fentanyl conformations, and how these conformations are being accommodated in the MOR binding pocket. We characterized the free energy landscape of fentanyl conformations with metadynamics simulations, and compared and evaluated several possible fentanyl binding conditions in the MOR with long-timescale molecular dynamics simulations. Our results indicate that the most preferred binding pose in the MOR binding pocket corresponds well with the global minimum on the energy landscape of fentanyl in the absence of the receptor, while the energy landscape can be reconfigured by modifying the fentanyl scaffold. The interactions with the receptor may stabilize a slightly unfavored fentanyl conformation in an alternative binding pose. By extending similar investigations to fentanyl analogs, our findings establish a structure-activity relationship of fentanyl binding at the MOR. In addition to providing a structural basis to understand the potential toxicity of the emerging NSOs, such insights will contribute to developing new, safer analgesics.

De Novo Design of Molecules with Low Hole Reorganization Energy Based on a Quarter-Million Molecule DFT Screen.

Materials exhibiting higher mobilities than conventional organic semiconducting materials such as fullerenes and fused thiophenes are in high demand for applications in printed electronics. To discover new molecules in the heteroacene family that might show improved hole mobility, three de novo design methods were applied. Machine learning (ML) models were generated based on previously calculated hole reorganization energies of a quarter million examples of heteroacenes, where the energies were calculated by applying density functional theory (DFT) and a massive cloud computing environment. The three generative methods applied were (1) the continuous space method, where molecular structures are converted into continuous variables by applying the variational autoencoder/decoder technique; (2) the method based on reinforcement learning of SMILES strings (the REINVENT method); and (3) the junction tree variational autoencoder method that directly generates molecular graphs. Among the three methods, the second and third methods succeeded in obtaining chemical structures whose DFT-calculated hole reorganization energy was lower than the lowest energy in the training dataset. This suggests that an extrapolative materials design protocol can be developed by applying generative modeling to a quantitative structure-property relationship (QSPR) utility function.

L-DOPA Dioxygenase Activity on 6-Substituted Dopamine Analogues.

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials or degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these potent catalysts. Here we report steady-state and pre-steady-state kinetic analysis of dopamine derivatives substituted at the 6-position as substrates of L-DOPA dioxygenase, and an analysis of that activity as a function of the electron-withdrawing nature of the substituent. Steady-state and pre-steady-state kinetic data demonstrate the dopamines are impaired in binding and catalysis with respect to the cosubstrate molecular oxygen, which likely afforded spectroscopic observation of an early reaction intermediate, the semiquinone of dopamine. The reaction pathway of dopamine in the pre-steady state is consistent with a nonproductive mode of binding of oxygen at the active site. Despite these limitations, L-DOPA dioxygenase is capable of binding all of the dopamine derivatives and catalyzing multiple turnovers of ring cleavage for dopamine, 6-bromodopamine, 6-carboxydopamine, and 6-cyanodopamine. 6-Nitrodopamine was a single-turnover substrate. The variety of substrates accepted by the enzyme is consistent with an interplay of factors, including the capacity of the active site to bind large, negatively charged groups at the 6-position and the overall oxidizability of each catecholamine, and is indicative of the utility of extradiol cleavage in semisynthetic and bioremediation applications.

Routine fixed-dose heparin vs. ACT-guided heparin administration for elective PCI and its influence on patient in-hospital outcome: a retrospective study.

OBJECTIVE: Activated clotting time (ACT)-based heparin dosing during percutaneous intervention (PCI) is recommended by Society guidelines. However, the relationship between ACT and outcome in the setting of elective PCI has not been sufficiently studied. We sought to evaluate the in-hospital outcome of patients undergoing elective PCI while receiving fixed-dose heparin without ACT measurement versus those with ACT-guided management. METHODS: This retrospective study included consecutive patients undergoing elective PCI in a single-center between 11/2015 and 12/2018. Patients were divided into two groups, depending on whether ACT was measured. Heparin-only anticoagulation and non-femoral procedures were allowed. Patient demographics, procedural data and in-hospital outcomes were collected. The primary outcome was in-hospital major adverse cardiovascular events (MACE), secondary (safety) outcomes were in-hospital definite stent thrombosis, Bleeding Academic Research Consortium bleeding, access-related complications (any) as well as peri-procedural complications. RESULTS: In total, 500 procedures were included in the study, 151 ACT and 349 fixed-dose. Patient demographics and medical history in both groups were well balanced, but those having ACTs were younger (63.2 ± 10.9 vs. 66.5 ± 11.3; P = 0.003) and less likely to have a history of coronary artery disease (74 vs. 82%; P = 0.032) or kidney failure. Procedural data were similar; however, total heparin dose and procedure length were higher in the ACT group (6232 ± 1388 vs.5032 ± 417 units; P < 0.001; 40.1 ± 14.0 vs. 30.3 ± 12.7 min; P < 0.001). Primary and secondary outcome events were rare and similar (MACE 1.1 vs. 1.3%; P = 0.86). CONCLUSIONS: A fixed-dose heparin injection (5000 IU) approach for elective PCI while omitting ACT offers slightly shortened procedural time and similar in-hospital safety profile.

A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells.

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a ß-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 µM, 300 µM, and 100 µM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 µM, 6.1 µM, and 3.4 µM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.

Glioma-Induced Alterations in Neuronal Activity and Neurovascular Coupling during Disease Progression.

Diffusely infiltrating gliomas are known to cause alterations in cortical function, vascular disruption, and seizures. These neurological complications present major clinical challenges, yet their underlying mechanisms and causal relationships to disease progression are poorly characterized. Here, we follow glioma progression in awake Thy1-GCaMP6f mice using in vivo wide-field optical mapping to monitor alterations in both neuronal activity and functional hemodynamics. The bilateral synchrony of spontaneous neuronal activity gradually decreases in glioma-infiltrated cortical regions, while neurovascular coupling becomes progressively disrupted compared to uninvolved cortex. Over time, mice develop diverse patterns of high amplitude discharges and eventually generalized seizures that appear to originate at the tumors' infiltrative margins. Interictal and seizure events exhibit positive neurovascular coupling in uninfiltrated cortex; however, glioma-infiltrated regions exhibit disrupted hemodynamic responses driving seizure-evoked hypoxia. These results reveal a landscape of complex physiological interactions occurring during glioma progression and present new opportunities for exploring novel biomarkers and therapeutic targets.

Complicated acute myocardial infarction with simultaneous occlusion of two coronary arteries.

Simultaneous occlusion of two coronary arteries in acute MI is infrequent and may be accompanied by cardiogenic shock. Prompt restoration of normal coronary flow can salvage the myocardium and decrease a possible risk of death from complications.

Publisher Correction: Regulation of protein kinase Cδ Nuclear Import and Apoptosis by Mechanistic Target of Rapamycin Complex-1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Massive Theoretical Screen of Hole Conducting Organic Materials in the Heteroacene Family by Using a Cloud-Computing Environment.

Materials exhibiting higher mobilities than conventional organic semiconducting materials such as fullerenes and fused thiophenes are in high demand for applications in printed electronics. To discover new molecules in the heteroacene family that might show improved charge mobility, a massive theoretical screen of hole conducting properties of molecules was performed by using a cloud-computing environment. Over 7 000 000 structures of fused furans, thiophenes and selenophenes were generated and 250 000 structures were randomly selected to perform density functional theory (DFT) calculations of hole reorganization energies. The lowest hole reorganization energy calculated was 0.0548 eV for a fused thioacene having 8 aromatics rings. Hole mobilities of compounds with the lowest 130 reorganization energy were further processed by applying combined DFT and molecular dynamics (MD) methods. The highest mobility calculated was 1.02 and 9.65 cm2/(V s) based on percolation and disorder theory, respectively, for compounds containing selenium atoms with 8 aromatic rings. These values are about 20 times higher than those for dinaphthothienothiophene (DNTT).

Reducing luteinizing hormone levels after ovariectomy improves spatial memory: Possible role of brain-derived neurotrophic factor.

Alzheimer's disease and other forms of cognitive decline are significantly more prevalent in post-menopausal women. Decreased estrogen levels, due to menopause or ovariectomy, may contribute to memory impairments and neurodegeneration. Another result of decreased estrogen levels is elevated luteinizing hormone (LH). Elevated LH after menopause/ovariectomy has been shown to impair cognition in both human and animal studies. Lowering LH levels rescues spatial memory in ovariectomized (ovx) rodents, yet the mechanisms of these effects are still unclear. Estrogens appear to exert some of their effects on memory by increasing levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. In these studies, we explored whether lowering LH may act by increasing BDNF. Ovx rats were treated with Antide, a gonadotropin releasing hormone receptor antagonist that lowers LH levels, or with estradiol. Both Antide and estradiol treatment enhanced spatial memory in ovx females. Both were found to be ineffective when a BDNF receptor antagonist was administered. Immunohistochemical analysis revealed that both Antide and estradiol increased BDNF expression in the hippocampus. Dendritic spine density on pyramidal cells in CA1 was unchanged by any treatment. These results provide evidence for a relationship between LH and BDNF in the hippocampus and demonstrate that estrogen-increasing and LH-lowering treatments may both require BDNF signaling in order to improve spatial memory.

Regulation of protein kinase Cδ Nuclear Import and Apoptosis by Mechanistic Target of Rapamycin Complex-1.

Inactivation of the protein complex 'mechanistic target of rapamycin complex 1' (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including 'protein kinase C delta' (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional 'target of rapamycin signaling' (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-ß. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-ß, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.