bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophagy and Glutamine Synthetase Induction Sustain ALL Blast Resistance to L‐Asparaginase in Human Bone Marrow Mesenchymal Stromal Cells
Authors: Chiu Martina , Bardelli Donatella , Taurino Giuseppe , Dander Erica , Bianchi Massimiliano G , Mirandola Prisco , Carubbi Cecilia , D'Amico Giovanna , Rizzari Carmelo , Bussolati Ovidio
Published: November 2023

L‐Asparaginase (ASNase) is one of the cornerstones of acute lymphoblastic leukaemia (ALL) treatment. Its antileukemic effect mainly relies on its ability to hydrolyse blood asparagine and glutamine. Asparagine depletion causes a severe nutritional stress in ALL blasts exhibiting low expression of asparagine synthetase (ASNS), which cannot, therefore, synthesize asparagine de novo. Conversely, given the role of glutamine as obliged nitrogen donor for asparagine synthesis, the glutaminolytic activity of ASNase is of relevance in the therapy of ASNS‐positive ALL blasts. It has been proposed that bone marrow mesenchymal stromal cells (MSCs) may support the resistance of ALL blasts to ASNase through the release of asparagine and glutamine. Given that no data are yet available on ASNase effects on MSCs themselves, aim of this work was to ascertain whether and how MSCs adapt to ASNase and if the inhibition of this adaptation reverts nutritional support to ALL blasts.

MSCs isolated from healthy donors and the ALL cell lines RS4;11 have been cultured in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FBS, 2 mM glutamine and antibiotics. Co‐cultures were established by adding RS4;11 cells to confluent monolayers of MSCs. ASNase from E. chrysanthemi was used at 1 U/ml, while glutamine synthetase (GS) was inhibited by 1 mM methionine‐L‐sulfoximine (MSO). Viability was measured through resazurin assay or flow cytometry. Autophagy was assessed by LC3 II detection in Western Blot.

MSCs were poorly sensitive to ASNase (IC50 > 10 U/ml) and, once treated with the enzyme, recovered their growth after a transient proliferative arrest. In MSCs, ASNase increased GS expression and activated a nutritional stress response marked by the phosphorylation of eIF2α. Moreover, ASNase inhibited mTORC1 activity, detected as the phosphorylation of p70S6K1, and, consequently, triggered autophagy. When GS was inhibited by MSO, the autophagic response was delayed, while CHOP was steadily induced and MSC growth was not rescued. When co‐cultured with MSCs, ALL blasts were effectively protected from ASNase‐induced cytotoxicity. The protective effect was markedly hindered when MSCs had been pre‐treated with ASNase and MSO. Our data show that the adaptation of MSCs to ASNase treatment through autophagy and GS induction is needed for ALL blast survival and may constitute an important therapeutic target.
The Anti‐migraine Effects of Delta‐9‐tetrahydrocannabinol in Female Rats are Mediated by CB1 Receptors
Authors: Kandasamy Ram , Dawson Cole T , Lee Andrea T , Morgan Michael M
Published: November 2023

Migraine affects 15% of the world's population, yet treatment options are limited by poor efficacy and adverse effects such as medication overuse headache. Anecdotal data suggest that marijuana may be an effective treatment for migraine. We tested this hypothesis by measuring whether administration of delta‐9‐tetrahydrocannabinol (THC), the psychoactive constituent of marijuana, has anti‐migraine effects in female rats. In addition, we administered cannabinoid receptor antagonists to determine whether these anti‐migraine effects are mediated by CB1 or CB2 receptors. Migraine pain was induced in female rats by microinjection of the TRPA1 agonist allyl isothiocyanate (AITC, 10%) onto the dura via a cannula implanted through the skull one week earlier. Migraine‐like pain was assessed by measuring changes in home cage wheel running. Rats were injected with either vehicle, the CB1 receptor antagonist SR141716 (1 mg/kg), or the CB2 receptor antagonist SR144528 (3.2 mg/kg) 30 min prior to simultaneous administration of AITC and vehicle or THC (0.32 mg/kg, i.p.). Microinjection of AITC caused a reduction in wheel running that lasted approximately 3 hours. Administration of THC attenuated the depression of wheel running caused by AITC administration. Administration of the CB1, but not the CB2, receptor antagonist attenuated the anti‐migraine effects of THC. Neither antagonist depressed wheel running in control animals. Taken together, these data indicate that the anti‐migraine effects of THC are mediated by CB1 receptors.

Support or Funding Information

This study was supported in part by State of Washington Initiative Measure No. 502 and NIH grant NS095097 (to MMM).
Effects of Cannabinoid Receptor Agonists on the Antinociceptive Effects of Oxycodone in Squirrel Monkeys
Authors: Withey Sarah Louise , Bergman Jack
Published: November 2023

Opioids remain the most commonly prescribed drug class for the management of moderate to severe pain and, often, high doses that have unwanted behavioral effects are required for effective analgesia. One strategy that has been proposed to avoid prescribing high doses of opioids is co‐administration of a cannabinoid such as Δ9‐tetrahydrocannabinol (THC) that, by providing its own analgesic effects, will reduce opioid dosage. Studies using the warm water tail withdrawal assay, which remains the most commonly used method for assessing antinociception across species, have provided encouraging data in support of this approach. Here, we further study the potential clinical advantage of this approach by determining how different cannabinoid agonists modify the effects of oxycodone on tail withdrawal latency and food‐maintained behavior (reinforcement density, response rate) in squirrel monkeys. Daily sessions were comprised of four 5‐min response components, each preceded by a long timeout during which cumulative doses of drugs (10‐min for oxycodone and 30‐min for THC or the nabilone derivative AM 7444) could be administered. During each component, the completion of a 10‐response fixed‐ratio in the presence of stimulus lights initiated food delivery and a 30‐sec short timeout during which stimulus lights were off and tail withdrawal latency was measured.

Results show that all three drugs produced dose related decreases on both measures of food‐maintained behavior but only oxycodone (0.032–0.56 mg/kg) and AM 7444 (0.0032–0.032 mg/kg) produced dose dependent increases in tail withdrawal latency. These antinociceptive effects were produced by doses of both drugs that did not greatly reduce reinforcement density and were more pronounced for oxycodone than the cannabinoid. Pretreatment with the highest dose of THC that did not eliminate responding (0.32 mg/kg) shifted the dose‐effect function for oxycodone's antinociceptive effects upward and leftward and its effects on food‐maintained behavior downward and leftward. Pretreatment with 0.0032 or 0.0056 mg/kg of AM7444 did not appreciably alter the antinociceptive effects of oxycodone whereas pretreatment with the higher dose (0.0056 mg/kg) markedly accentuated the effects of oxycodone on food‐maintained behavior.

These data support previous findings that cannabinoid pretreatment can heighten the antinociceptive effects of morphine‐like opioids but indicate also that cannabinoid pretreatment may exacerbate their behaviorally disruptive effects. Thus, this approach to pain management, while effective, may not be appropriate for all therapeutic settings.
Anti‐Nociceptive Effect of Chrysin in Partial Sciatic Nerve Ligation Model
Authors: Sujatha Dodoala , Begum Nazeef , Prasad KVSRG
Published: November 2023

Chrysin, a natural polyphenol is acclaimed for its anti‐tumor, anti‐oxidant, anti‐allergic and anti‐inflammatory activities. Few reports were available to support its use against neuropathic pain. The current investigation was designed to study and characterize the anti‐nociceptive effects of chrysin in partial sciatic nerve ligation (PSNL) model induced in male Wistar rats. We used capsaicin‐induced nociception test to verify whether chysin acts via transient receptor potential vanilloid (TRPV1) receptors, as in silico studies in our laboratory has shown good binding affinity of chrysin at the vanilloid binding site of the receptor. All the animal protocols were approved by Institutional Animal Ethical Committee (Regd No.1677/PO/RE/S/2012/CPCSEA). The rats received treatment with vehicle (PSNL control), gabapentin (Standard, 25 mg/kg, p.o) or chrysin (100 and 150 mg/kg, p.o.) for 28 days. The various behavioral and biochemical parameters were determined at regular time intervals and histopathological parameters were evaluated at the end of the study. The data was presented as mean ± SEM and one‐factor ANOVA followed by post hoc analysis using Bonferroni test. Chronic treatment with chrysin (100 and 150 mg/kg) significantly and dose‐dependently improved (p < 0.05) the nociceptive threshold, measured as allodynia, thermal hyperalgesia and mechanical hyperalgesia using standard protocols. The decreased activity of superoxide dismutase, catalase, reduced glutathione, membrane bound inorganic phosphate (Na+/K+ Atpases) and increased lipid peroxidation and Ca2+ Atpases were significantly (p < 0.05) restored by chrysin treatment. Histological aberrations induced after PSNL in sciatic nerve were restored by treatment with chrysin (100 and 150 mg/kg, p.o). Intraplantar injection of capsaicin induced flinching and licking response serves as an index of nociception and was observed in all the treated groups. Chrysin treated groups shown marked reduction in both flinching and licking responses when compared to the control group. The biochemical, behavioural and morphological studies clearly revealed that chrysin protects against PSNL induced neuropathic pain in a dose‐response manner which can be attributed to its multiple mode of action on neuropathic pain targets.

Support or Funding Information

The work was funded by Sri Padmavati Mahila Visvavidyalayam under the support given for the post graduate research work.
Effect of Acute and Chronic Stress on Somatic Pain Sensitivity under Circumstances of Indomethacin‐Induced Gastrointestinal Injury in Conscious Rats
Authors: Filaretova Ludmila , Bagaeva Taniana , Iarushkina Natalia
Published: November 2023

Nonsteroidal anti‐inflammatory drugs (NSAIDs) such as indomethacin (IM) are commonly used; however their use is complicated by the side effects on the gastrointestinal tract. Gastrointestinal injury triggers the changes in visceral as well as somatic pain sensitivity in rats. Here we studied the effect of acute and chronic stress on somatic pain sensitivity under circumstances of IM‐induced gastric injury in conscious rats. Gastric erosion was caused by a single IM injection (35 mg/kg, sc) in preliminary (24 h) fasted rats. Somatic pain sensitivity was assessed by tail flick test. The animals were habituated for tail flick test for 7 days and examined for baseline tail flick latency on day 8. Then, animals were exposed to IM administration with acute or chronic stress or without stress. Cold‐restraint stress at 10 °C for 30 min and keeping at room temperature 1h before IM was used as acute stress. For chronic stress the rats were subjected to unpredictable stress of various modalities for 14 days daily and injected by IM on day 15. Tail flick latencies were measured after stress (before IM) and 4 h after IM administration. Gastric mucosa injury, plasma corticosterone levels, blood pressure, heart rate, adrenal and thymus weight were also measured. IM caused gastric injury 4h after its administration. Acute or chronic stress by itself did not influence gastric mucosa, but its action manifested under circumstances of IM‐induced gastric injury. Acute stress attenuated the ulcerogenic effect of IM on gastric mucosa, but chronic stress potentiated it. The formation of gastric erosion was accompanied by an increase in tail flick latencies (somatic hypoalgesia). We did not observe any changes in tail flick latencies 1h after cold‐restaint stress. However, acute stress prevented somatic hypoalgesia induced by gastric injury. In contrast to acute stress, chronic stress attenuated tail flick latencies before IM administration, but did not affect somatic hypoalgesia. The data obtained suggests that chronic stress aggravates the ulcerogenic action of IM on gastric mucosa that is accompanied by an increase in somatic pain sensitivity, whereas acute stress plays the protective role in the maintenance of integrity of gastric mucosa and preventing of pain abnormalities induced by pathological process in stomach.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790.
ARHGEF10 Knockout Mice Exhibit Autism‐Like Behavior and Altered Pain Sensation
Authors: Lu DaiHua , Liou Houng‐Chi , Liao Hsiao‐Mei , Chen Chia‐Hsiang , Gau Susan Shur‐Fen , Fu Wen‐Mei
Published: November 2023

Autism spectrum disorder (ASD) is a complex neurodevelopment disorder which can cause social, communication and development problems. Genetics certainly play a crucial role in ASD, however, how these different genes affect autistic‐like behavior is still unknown. Our previous CNV study discovered several novel CNVs associated ASD. Among the deleted CNV regions, a missense mutation of the ARHGEF10 is found to be the contributory factor in central nervous system diseases. Here we identified a new autism‐related gene, ARHGEF10, that codes a Rho guanine exchange factor 10 which is a member of guanine nucleotide exchange factor (GEF) family and activates small GTPases but preferentially for RhoA. To follow these CNV findings and gain deep insight into the pathogenesis of autism, we generated a ARHGEF10 knockout mice based on the identification of the patient with 8p23 terminal deletion.

We found that the mice lacking ARHGEF10 displayed hyper‐locomotor activity in open field and alternated anxiety behavior in plus maze test. In addition, ARHGEF10 knockout mice showed impaired social interaction and social recognition in three‐chamber test. Moreover, the response to plantar and formalin test was reduced in ARHGEF10 knockout mice. However, knockout ARHGEF10 did not affect spatial learning memory and sensory motor gating.

Our findings demonstrate that ARHGEF10 knockout mice displayed key symptoms of autism spectrum condition in human including decreased social interaction and increase spontaneous activities, reduced pain sensation. These results, together with the finding of CNVs in ASD patient, demonstrated that ARHGEF10 may be a candidate gene of autism.

Support or Funding Information

This work was supported by the Ministry of Science and Technology grants, 105‐2321‐B‐002‐020, Taiwan.
&[Beta]ETA‐CYCLODEXTRIN‐COMPLEXED WITH LIPPIA GRATA LEAVES ESSENTIAL OIL REDUCES HYPERALGESIA IN MICE
Authors: Quintans Jullyana Siqueira , Siqueira‐Lima Pollyana , Menezes Erik Pereira , Filho Heitor Gomes Araújo , Araujo Adriano , Rezende Marilia M , Amarante Ruthy Kellen Lima , de Souza Barreto Rosana , Lucchese Angelica , Quintans Lucindo José
Published: November 2023

Lippia grata Schauer is a medicinal plant native of the semi‐arid area of Northeastern Brazil used in folk medicine as analgesic, anti‐inflammatory and antispasmodic. The leaf essential oil (EO) obtained from L. grata is rich in camphor, (E)‐caryophyllene, bicyclogemacrene, camphene and borneol which has already demonstrated analgesic profile. The pharmacological uses of EO could be limited by short half‐life and insolubility in water but β‐cyclodextrin (CD) could be an effective tool to improve this. Thus, we assessed the pharmacological profile of EO and EO complexed with CD (CDEO) in a model of neuropathic pain induced by injury of the sciatic nerve and in an inflammatory model (pleurisy model) in mice. All experiments were approved by the Animal and Use Committee of the Federal University of Sergipe (CEPA/UFS# 65/2015). Mice were pretreated with EO and CDEO (24 mg/kg, orally route) or vehicle at 7 days after surgery and 1h before carrageenan pleural injection. The treatment with EO and CDEO were able to promote antihyperalgesic effect in mechanical (analgesymeter) and increase latency for thermal stimulation (hot plate test) (p< 0,001) when compared with control group. The treatment with EO or CDEO were effective in reducing the leukocyte influx to pleural cavity and also reduced levels (p < 0,001) of IL‐1beta and TNF‐alpha in the pleural lavage. Moreover, no motor abnormalities were observed after treatment with EO and CDEO. Our findings demonstrated that EO and CDEO possess an analgesic and anti‐inflammatory profile in neuropathic pain and inflammatory models respectively, which seems to be involved by reduction of pro‐inflammatory cytokines.

Support or Funding Information

CNPq, CAPES, FAPITEC‐SE, FINEP
Differing Roles of S1P Receptor Adaptation in FTY720‐Induced Antinociception in Acute Versus Neuropathic Pain
Authors: Selley Dana E. , Wilkerson Jenny L. , Welch Sandra P. , Donvito Giulia , Lichtman Aron H. , Sim‐Selley Laura J.
Published: November 2023

Chronic neuropathic pain is difficult to treat because currently available medications have limited effectiveness and/or serious side effects, including abuse liability. Therefore, new approaches are needed to treat this condition. The immunomodulatory pro‐drug FTY720, which when phosphorylated acts as an agonist for G‐protein‐coupled S1P receptors (S1PR) including S1PR1, 3, 4 an 5, has been shown to be antinociceptive in rodent models of neuropathic pain. S1P and S1PR agonists also produce antinociception in acute thermal pain models in rodents. Here we show that FTY720 produces both acute thermal antinociception and alleviation of allodynia in the chronic constriction injury (CCI) model of neuropathic pain in mice. These effects were both dose‐dependent and mimicked by the S1PR1 selective agonist CYM‐5442. Importantly, repeated administration of FTY720 for one week produced tolerance to the acute thermal antinociceptive effect of the drug, but did not produce tolerance to its anti‐allodynic effect in the CCI model. Examination of S1PR‐stimulated G‐protein activation using agonist‐stimulated [35S]GTPγS autoradiography revealed apparent desensitization of G‐protein activation stimulated by S1P or the S1PR1 agonist SEW2871 throughout the brain. Similar results were seen in membranes prepared from the spinal cord, whereby S1P concentration‐effect curves showed that the Emax value of S1PR‐stimulated [35S]GTPγS binding was greatly reduced in repeated FTY720‐treated mice. These results suggest that S1PR1 is the primary target of FTY720 in alleviation of both acute thermal pain and chronic neuropathic pain induced by nerve injury. Furthermore, the finding that tolerance develops to the antinociceptive effect of FTY720 in acute thermal pain but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these two models. Because S1PR1 signaling was desensitized throughout the CNS after repeated FTY720 treatment, it is possible that that this loss in receptor signaling could be responsible for both tolerance to acute thermal antinociception and for the anti‐allodynic action of FTY720 in the CCI model. Therefore, activation of S1PR1 could be responsible for acute thermal antinociception, whereas desensitization of S1PR1 could contribute to the anti‐allodynic effect in neuropathic pain by producing functional antagonism of pro‐nociceptive S1PR1 signaling.

Support or Funding Information

Supported by NIH grant R01‐NS093990 from the National Institute of Neurological Disorders and Stroke.
The Transition from Stochastic to Collective Activation of Nicotinic Acetylcholine Receptors Facilitates Information Transmission Within in vitro Hippocampal Networks
Authors: Djemil Sarra , Chen Xin , Dzakpasu Rhonda
Published: November 2023

Although modulatory in nature, nicotinic acetylcholine receptors (nAChRs) play a major role in the central nervous system, making them one of the most studied receptor class. While both activation and desensitization are phenomena associated with the functions of individual receptors, their effects will clearly manifest on the network/circuit spatial scale. In this study, nicotine was used to investigate the impact of activation and desensitization of nAChRs on cultured hippocampal networks. We assess network dynamics via the recording of extracellular action potentials using a microelectrode array system (MEA). We bath applied either a single dose or two successive doses of increasing concentration of nicotine. We hypothesize that a high concentration of nicotine will facilitate information transmission but pre‐incubation of the network with a low concentration of nicotine would attenuate facilitation. We show that the low concentration has minimal effects on network activity and we call this a network‐desensitized state. A high concentration significantly impacted both spiking and bursting activity and we call this a network‐activated state. Interestingly, a low concentration pre‐incubation of nicotine blocked or significantly diminished activity when followed by a high concentration. Lastly, network synchronization as well as network efficiency increased with the large nicotine concentration. This suggests that although fleeting, the process of global receptor activation has a longer lasting impact on the network than a stochastic receptor activation.

Support or Funding Information

National Science Foundation grant no. PHY‐1205919
Pharmacologic Inhibition and Genetic Elimination of p38α MAPK Normalizes Serotonergic System Alterations and Social Behavior Deficits in the SERT Ala56 Mouse Model of Autism Spectrum Disorder
Authors: Robson Matthew J. , Quinlan Meagan A. , Veenstra‐VanderWeele Jeremy , Watterson D. Martin , Blakely Randy D.
Published: November 2023

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder exhibiting language and communication deficits, perturbations in social interactions, and rigid, repetitive behaviors. Currently, it is estimated that nearly 1 in 65 children lie on the diagnosable spectrum of ASD. Amplifying this problem, there are no FDA approved medications that alleviate the core symptoms of the disorder. Genetic variation in the SLC6A4 gene that encodes the serotonin (5‐HT) transporter (SERT) has been associated with both ASD and hyperserotonemia, a well‐replicated ASD biomarker. Mice that express the ASD‐associated SERT coding variant Ala56 exhibit hyperserotonemia, elevated CNS 5‐HT clearance, a hypersensitivity of 5‐HT1A and 5‐HT2A receptors and multiple ASD‐like behavioral phenotypes including alterations in social interactions. SERT protein in Ala56 mice exhibit hyperphosphorylation which can be normalized by inhibition of p38 MAPK. Of the four known isoforms of p38 MAPK (α, β, γ, and δ), work has revealed p38α MAPK as the primary isoform responsible for SERT regulation. We hypothesized that selective, CNS penetrant, p38α MAPK inhibitors such as the recently developed MW108 or MW150 might attenuate one or more ASD‐like phenotypes in the SERT Ala56 model. Treatment with MW108 (10mg/kg, QD × 1 week) or MW150 (5 mg/kg, QD × 1 week) normalized SERT Ala56‐mediated hypersensitivity of 5‐HT1A and 5‐HT2A receptors. Further chronic treatment with MW150 normalized SERT Ala56‐mediated increases in hippocampal 5‐HT clearance. Chronic MW108 or MW150 treatment normalized SERT Ala56‐mediated social deficits in the tube test without concomitant changes in midbrain SERT protein levels or changes in tissue levels of monoamine neurotransmitters or their metabolites. Acute MW150 (5 mg/kg) treatment lacked effect on 5‐HT receptor and social behavioral indices. Moreover, genetic elimination of p38α MAPK in 5‐HT neurons of SERT Ala56 mice resulted in the normalization of 5‐HT2A receptor hypersensitivity and social deficits in the tube test as compared to their SERT Ala56 counterparts. Our findings support the conclusion that ongoing p38α MAPK signaling sustains the hyperfunction of SERT Ala56 and subsequent alterations in 5‐HT signaling and behavior. These are the first studies to provide pharmacologic and genetic evidence that targeting p38α MAPK signaling may be a potential therapeutic path for the treatment of ASD.

Support or Funding Information

This work was supported by NIH grants MH078028, MH094527, NS007491 and U01AG043415. Additional support was provided by the Simons Foundation Autism Research Initiative, the PhRMA Foundation and the Brain & Behavior Research Foundation.
Expression and functional characterization of PEA‐15 within the suprachiasmatic nucleus
Authors: Wheaton Kelin , Queiroz Lucas Sales , Sullivan Kyle , Obrietan Karl , Hoyt Kari
Published: November 2023

The circadian (24 hour) timing system regulates the functional features of most, if not all, physiological process. As such, disruption of the circadian timing system has been shown to have profound negative effects on health, cognition, and to contribute to an array of disease states. Central to the mammalian circadian timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. This brain structure (often referred to as the ‘master clock’) generates timing cues that coordinates the phasing and amplitude of oscillator populations found throughout the brain, and in peripheral organ systems, such as the liver, lungs and heart. Notably, phasing of the SCN is tightly regulated by photic input signals that are relayed from the retina. The p44/42 mitogen‐activated protein kinase (MAPK) pathway has been shown to be a critical component of the SCN timing system and to function as a key route through which the photic input resets the clock. Here, we investigated cellular mechanisms that regulate ERK (the key effector kinase of the MAPK pathway) phosphorylation and its cellular localization within the SCN. To this end, we examined the expression and function of phosphoprotein enhanced in astrocytes (PEA‐15), a scaffold protein that has been shown to regulate both the activation state of ERK and its subcellular localization. A combination of immunohistochemical labeling and Western analysis methods revealed that PEA‐15 is highly expressed within the mouse SCN. Furthermore, expression profiling detected a significant circadian oscillation in PEA‐15 within the SCN; interestingly, the peak in PEA‐15 expression was antiphase to the well‐characterized circadian oscillation in ERK activity. Photic stimulation of mice led to a significant increase in PEA‐15 phosphorylation at serine‐104, an event that is responsible for the dissociation of ERK from PEA‐15. Consistent with this, co‐immunoprecipitation of ERK and PEA‐15 from SCN lysates revealed that PEA‐15 is directly bound to ERK, and that photic stimulation triggers the dissociation of ERK from PEA‐15. Together, these data reveal that, 1) PEA‐15 is expressed in the SCN, 2) PEA‐15 expression is under the control of the circadian timing system, and 3) PEA‐15 regulates signaling via the MAPK pathway. Given that MAPK signaling is a key regulator of the clock timing system, the data reported here raise the prospect that PEA‐15 could play a key role in modulating the functionality of the SCN timing system. Ongoing work is focused on an examination of potential mechanism by which PEA‐15 affects the timing properties of the SCN.

Support or Funding Information

This work was supported by NIH MH103361.
Evaluating the Therapeutic Potential of Serotonin Modulators in Scn1a‐derived Epilepsy
Authors: Wong Jennifer C. , Murnane Kevin S. , Blough Bruce E. , Escayg Andrew
Published: November 2023

De novo loss‐of‐function mutations in the voltage‐gated sodium channel SCN1A (encoding Nav1.1) are the main cause of Dravet syndrome (DS), a catastrophic early‐life encephalopathy associated with prolonged and recurrent early‐life febrile seizures (FSs), refractory afebrile epilepsy, cognitive and behavioral deficits, and a 15–20% mortality rate. SCN1A mutations also lead to genetic epilepsy with febrile seizures plus (GEFS+), which is an inherited disorder characterized by early‐life FSs and the development of a wide range of adult epilepsy subtypes. Current antiepileptic drugs (AEDs) often fail to provide adequate protection against the severe seizures and neuropsychiatric comorbidities that occur in patients with SCN1A mutations. Furthermore, almost a third of epilepsy patients do not achieve adequate seizure control, highlighting the urgent need to develop multimodal treatments that can effectively mitigate the broad spectrum of clinical features associated with refractory epilepsies, while minimizing unwanted side effects. Heterozygous Scn1a knockout mice (Scn1a+/−, a model of DS) and heterozygous Scn1a knock‐in mice expressing the human SCN1A epilepsy mutation R1648H (Scn1aRH/+, a model of GEFS+) recapitulate many of the clinical features found in patients with DS and GEFS+, and thus provide an important opportunity to develop more efficacious AEDs.

There is increasing evidence to suggest a therapeutic role of increased serotonin (5HT) levels in the treatment of refractory epilepsy. Therefore, we evaluated, using mouse models of SCN1A dysfunction, the therapeutic potential of fenfluramine, which has recently been used as an add‐on therapy in a small number of patients with DS. In addition, the efficacy of two serotonin receptor agonists selective for 5HT2A (DOI) and 5HT2C (WAY163909) were also examined. We first generated a dose‐response curve for each compound in male CF1 mice using the well‐established 6 Hz seizure induction paradigm. Next, we found that fenfluramine (17 mg/kg) increased the latency to the first pentylenetetrazole (PTZ)‐induced generalized tonic‐clonic seizure in Scn1a mutant mice. The 5HT2A agonist (3 and 5.6 mg/kg) also increased resistance to PTZ‐induced seizures and electrically‐induced seizures (6 Hz and MES) in Scn1a mutant mice. Taken together, this study provides justification for additional investigation into the benefit of increasing serotonin signaling as a novel therapeutic strategy in DS and GEFS+, and more broadly, in other forms of refractory epilepsy.

Support or Funding Information

2T32NS00748016 (JCW), Blue Cotinga LLC
Increasing KCC2 function as a strategy for the treatment of seizures
Authors: Moore Yvonne , Deeb Tarek , Moss Stephen
Published: November 2023

Fast synaptic inhibition is mediated by GABAA receptors, which allow Cl− entry into neurons. This occurs due to an inwardly directed Cl− gradient established by KCC2, which extrudes Cl− from neurons. KCC2 dysfunction is a genetic cause of epilepsy and its function is downregulated in many patients with seizure‐related disorders, suggesting a broader role for KCC2 dysfunction in epilepsy. Consequently, it has been hypothesized that increasing KCC2 function would be beneficial in treating epilepsy patients. However, due to the lack of pharmacological activators of KCC2, it has not yet been possible to test this hypothesis.

Phosphorylation of KCC2 at residues threonine 906 and threonine 1007 (T906/T1007) reduces KCC2 function. We have created a KCC2‐T906/T1007‐Alanine knock‐in mouse in an attempt to increase KCC2 function in the brain. Basal EGABA values are substantially hyperpolarized in these mice and the rate in which Cl− can be extruded from neurons is increased. This confirms we have developed the first KCC2‐gain‐of‐function mouse model.

In three different in vitro models of seizure‐like‐activity, the KCC2‐gain‐of‐function mice showed a prominent reduction in seizure severity. This study provides direct evidence that increasing KCC2 function is a valid therapeutic strategy to alleviate the burden of epilepsy.
Inhibition of Membrane‐Induced Alpha‐Synuclein (aSyn) Aggregation: A Strategy to Interfere with aSyn Neurotoxicity in Parkinson's Disease (PD)
Authors: Chandran Aswathy , Ysselstein Daniel , Dehay Benjamin , Costantino Isabel , McCabe George P , Frosch Matthew , George Julia M , Bezard Erwan , Rochet Jean‐Christophe
Published: November 2023

Oligomerization of the presynaptic protein aSyn is thought to play a key role in the pathogenesis of PD. aSyn exists in a number of conformations, including a membrane‐bound state likely involved in regulating synaptic vesicle trafficking. aSyn interacts with anionic phospholipid vesicles by forming an amphipathic α‐helix of various lengths. We hypothesize that disruption of interactions between aSyn and phospholipid membranes leads to a shift to a short‐helix, lipid‐bound form, which is more susceptible to forming toxic aSyn oligomers due to exposure of the protein's central hydrophobic region. Therefore, stabilization of the long‐helix form of lipid‐bound aSyn should prevent membrane‐induced aggregation and alleviate aSyn neurotoxicity. To address this hypothesis, endosulfine alpha (ENSA), a protein that interacts selectively with membrane‐bound aSyn, and a series of peptides identified as interactors of lipid‐bound aSyn via phage display screening were characterized in terms of their effects on membrane‐induced aSyn aggregation, aSyn‐mediated membrane permeabilization, and aSyn neurotoxicity in primary midbrain cultures. We found that A30P and G51D, two aSyn variants with disrupted membrane interactions, had an increased ability to form SDS‐resistant aggregates at the membrane surface and to elicit neurotoxicity than wild type (WT) aSyn, and both triggered the disruption of dye‐loaded vesicles under conditions identical to those that promoted membrane‐induced aggregation. WT ENSA (but not the non‐aSyn‐binding S109E variant) and a subset of phage display peptides attenuated aSyn self‐assembly at the membrane, vesicle disruption, and aSyn neurotoxicity. Intriguingly, ENSA was found to be down‐regulated in the brains of patients with dementia with Lewy bodies or PD versus non‐diseased individuals. Current efforts are focused on characterizing the aggregation of aSyn variants in biological membranes and monitoring aSyn‐mediated vesicle permeabilization in neuronal cells, with the ultimate aim of elucidating neuroprotective mechanisms of ENSA and aSyn‐binding peptides in cellular models relevant to PD. Our findings strongly support the idea that membrane‐induced aSyn aggregation plays a critical role in aSyn neurotoxicity, and they suggest that targeting this process is a viable therapeutic strategy for PD.

Support or Funding Information

Branfman Family Foundation, Indiana CTSI Core Pilot Grant, Indiana CTSI Collaboration in Translational Research Grant, Richard F. Borch Research Enhancement Award
The Neurotoxic Effect of Manganese on Dopamine Post‐Synaptic Receptors Are Reversed by Taurine
Authors: Loney‐Walsh Kurt , Dubose Loren , Catapane Edward J , Carroll Margaret A
Published: November 2023

Manganese (Mn), a neurotoxin causing the human disease Manganism, a Parkinson's‐like syndrome, disrupts dopamine neurotransmission in the brain. The neurotoxic mechanism of action is not fully resolved. Reports postulate manganese toxicity is more related to post‐synaptic dopamine D2 receptors (D2DR) dysfunction, than degeneration of dopamine neurons. Gill lateral ciliated cells of Crassostrea virginica are innervated by cilio‐inhibitory dopamine nerves via the branchial nerve and have D2DR type post‐synaptic receptors. Our previous physiological work found Mn treatments block the cilio‐inhibitory effect of dopamine on gill lateral cells and our immunohistofluorescence work with FITC‐labeled 2° antibodies against anti‐D2DR showed Mn treatments reduced FITC fluorescent intensity in gill lateral cells. The results suggest that either Mn toxicity causes a loss of D2DR number or a structural disruption of receptor integrity. Currently there is no effective clinical treatment for Manganism. There are reports the drug p‐aminosalicylic acid (PAS) alleviates some of the symptoms and our physiological and immunohistofluorescence studies with C. virginica showed PAS reversed the toxic action of Mn on the animals' dopaminergic system. Recently the amino acid taurine has been shown to alleviate symptoms in a number of neurodegenerative diseases. In the present study we hypothesize taurine would reverse the neurotoxic effect of Mn on gill lateral cell D2DR in C. virginica. To test this C. virginica were treated for 3 or 5 days with Mn (500 μM) followed by 3 or 5 days with taurine (500 μM), respectively. Gills were excised and treated for immunohistofluorescence as previously done. Briefly, gills were fixed in paraformaldehyde, exposed to rabbit anti‐D2DR 1° antibodies followed by FITC‐labeled goat anti‐rabbit 2° antibodies, paraffin embedded, sectioned at 10 μm and visualized on a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software from NSF. Results show fluorescence intensity in gill cells of animals treated with Mn had a statistically significant decrease in fluorescence, 66% less than non‐Mn treated controls. Animals treated with 3 days of Mn followed by 3 days of taurine had gill cells showing only an 18% reduction in fluorescence intensity compared to controls. Animals treated 5 days with Mn followed by 5 days with or without taurine gave similar results. The study indicates taurine was able to reverse the Mn induced loss of post‐synaptic D2DR fluorescence similarly to what occurred with PAS treatments. The neuroprotective mechanism of taurine needs to be further investigated. This immunohistological study suggests taurine may be a promising therapeutic agent in the treatment of Manganism in humans.

Support or Funding Information

This work was supported in part by PSC‐CUNY grant 690340047, 2R25GM06003 of the Bridge Program of NIGMS and a Carnegie Foundation award.
Downregulation of GRP78 chaperone protein triggers pro‐apoptotic CHOP signaling cascade in acute intracerebral hemorrhage rats
Authors: Liew Hock‐Kean , Chen Po‐Kong , Peng Hsiao‐Fen , Chen Jung , Lin Peter Bor‐Chian , Wang Po‐Kai , Pang Cheng‐Yoong , Chen Tsung‐Ying
Published: November 2023

Endoplasmic reticulum (ER) stress triggered‐apoptosis might play important role in many neurological disorders, leading to brain damage consequently. Glucose regulated protein (GRP) 78 or Bip, serves as a master controller in stabilizing the unfolded proteins resulted from various stimuli in the ER and thus reduces ER stress. Although upregulation of GRP78/Bip possesses neuroprotective effect in ischemic stroke animals, its role in the most deadly type of stroke ‐ intracerebral hemorrhage (ICH), remains unclear. The aim of this study work was to study the role of GRP78/Bip during ICH injury.

ICH were induced in rats by intrastriatal infusion of 0.2 U collagenase VII‐S. The neurological severity was evaluated before and at 1‐, 3‐ and 7‐days after ICH. At 0, 1, 3, 6, 24, and 72 hours, brains were recovered for hematoma volume measurement, and molecular biological or histological studies.

Our results demonstrated that GRP78/Bip rapidly decreased in the ipsilateral striata during 1 to 3 hours post‐ICH, and gradually restored to the basal level at 24 hours post‐ICH. The downregulation of GRP78/Bip was associated with the severity of the hemorrhagic volume. Reversed PCR of GRP78/Bip indicated the decrease of GRP78/Bip was not due to the decreased expression of GRP78/Bip gene, but was the result of increased ubiquitin‐mediated degradation. The decrease of GRP78/Bip triggers the pro‐apoptotic CHOP to be activated during the 24 to 72 hours post‐ICH. The activation of CHOP was associated with the volume of the hematoma and the severity of the ICH‐induced brain injury. Thus, the decrease of GRP78/Bip at the early phase was correlated with the severity of ICH injury, and treatment targeting on regulating Bip/GRP78 might has a clinical potential in reducing ICH‐induced brain injury.
Mechanisms and Regulation of PLCβ and its Effects on Membrane Adsorption
Authors: Hudson Brianna N , Hyun Seok‐Hee , Thompson David H , Lyon Angeline M
Published: November 2023

Phospholipase C (PLC) enzymes are peripheral membrane proteins that are activated by the Gq subfamily of G‐protein coupled receptors. The enzymes hydrolyze the membrane component phosphatidylinositol‐4, 5‐bisphosphate (PIP2) into two potent second messengers, inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG), in response to diverse stimuli. PLCβ enzymes are required for normal cardiovascular function and increased expression is associated with arrhythmias, hypertrophy, and heart failure. Previous studies have shown the C‐terminal domains of PLCβ contribute to its regulation and membrane association. However, there is no structural information on how the membrane and C‐terminal domains contribute to PLCβ regulation. We seek to understand how the membrane enhances association and promotes conformational changes within PLCβ that increase catalytic activity. Beginning with model membrane systems, we are applying atomic force microscopy and complementary biochemical assays to evaluate the role of the C‐terminal regulatory domains in membrane adsorption and activity. We are also investigating how the physical and chemical properties of the membrane itself, e.g. surface charge, fluidity, and composition, regulate PLCβ adsorption and activation. These studies provide the first structure‐based approach to understanding how the cell membrane itself regulates the activity of this essential effector enzyme.

Support or Funding Information

The Purdue Doctoral Fellowship
RhoA Regulates PINK1 Accumulation and Mitophagy through PKD Activation
Authors: Tripathi Raghav , Tan‐Sah Valerie P , Brown Joan Heller , Miyamoto Shigeki
Published: November 2023

RhoA is a small GTPase that regulates myriad cellular events, including adhesion, migration, invasion, cell cycle progression, gene expression, and cellular protection. Our previous research demonstrated that expression of RhoA in the heart protects cardiomyoctes from ischemia/reperfusion injury. RhoA is activated by various GPCRs, including those for sphingosine‐1‐phosphate (S1P); activation of RhoA contributes to S1P‐mediated cardioprotection. We recently determined that RhoA activation in the heart increases mitochondrial autophagy (mitophagy), a process that eliminates compromised mitochondria and thereby diminishes induction of mitochondrial death pathways. The involvement of RhoA induced mitophagy in the cardioprotective response to RhoA has not been examined. The accumulation of PTEN‐induced putative kinase 1 (PINK1) at damaged mitochondria plays an important role in induction of mitophagy. Increasing RhoA expression through adenovirus in neonatal rat ventricular myocytes (NRVMs), or in the mouse heart by AAV9 RhoA injection, leads to increased PINK1 expression at mitochondria. Importantly, this also increases Parkin recruitment to mitochondria, ubiquitination of mitochondrial proteins, and induction of autophagosomes containing mitochondria. Interestingly, there is no concomitant mitochondrial membrane depolarization associated with these responses. qRT‐PCR analysis also showed no difference in PINK1 mRNA levels in control and RhoA‐expressing cells. Degradation of exogenously expressed PINK1 protein in the presence of cycloheximide was, however, greatly inhibited by RhoA expression. ROCK is one of the most established RhoA downstream effectors, but inhibition of ROCK did not affect PINK1 accumulation induced by RhoA. We previously showed that RhoA activates PKD to confer cardioprotection. Studies using siRNA‐mediated knockdown of PKD or pharmacological inhibition showed that this largely attenuated RhoA mediated PINK1 stabilization. These results suggest that RhoA increases PINK1 levels at mitochondria by preventing its degradation through PKD and in a mitochondrial membrane depolarization‐independent fashion. We plan to further investigate the functional significance of RhoA‐dependent PINK1 induction and mitophagy in cardioprotection against sustained ischemia in vivo (e.g. myocardial infarction).

Support or Funding Information

This work was supported by the ASPET SURF Program and NIH HL028143.
Inhibition of Heat Shock Protein 90 Reduces Opioid‐Induced Anti‐Nociception by Blocking ERK Mitogen Activated Protein Kinase Signaling in Mouse Brain
Authors: Lei Wei , Mullen Nate , Brann Courtney , Cormier James , Bilsky Edward , Streicher John Michael
Published: November 2023

Opioid receptor signaling is one of the primary targets for drug discovery for chronic pain. Heat shock protein 90 (Hsp90) is a highly expressed and ubiquitous regulator for various signaling pathways and proteins. Only 2 Hsp90 studies have been performed in pain models, finding that Hsp90 inhibition reduced diabetic sensory hypoalgesia and inflammatory pain, and a further 2 studies found that Hsp90 inhibition reduced opioid dependence in cell and mouse models. However, these are only a few studies, and the inhibitors were given acutely (<3 hours), which may not be a good model for chronic pain treatment. Hsp90 thus represents an understudied mechanism for the regulation of opioid‐induced analgesia and side effects. To study Hsp90 regulation in mouse brain, we established a model of 24‐hour Hsp90 inhibition, to more closely model chronic pain treatment. This was performed in CD‐1 mice by intracerebroventricular (icv) injection of the selective Hsp90 inhibitor 17‐AAG. The 17‐AAG dose used for all studies was established by dose/response experiments. We found that Hsp90 inhibition strongly blocked morphine‐induced anti‐nociception to mechanical allodynia (Von Frey) in a post‐surgical paw incision model (79.3%), but only modestly blocked (21%) DAMGO‐induced anti‐nociception in a naïve tail flick model. Hsp90 inhibition also worsened morphine‐induced acute and chronic dependence. These changes were not due to sedation, motor effects, or other non‐opioid mechanisms, as 17‐AAG treatment caused no decrease in performance in the rotarod test. Seeking a mechanism for these changes, we found that Hsp90 inhibition increased Hsp70 expression and blocked ERK MAPK activation in response to DAMGO in the periaqueductal grey (PAG) and caudal brain stem. We modeled these signaling changes by icv injection of the selective ERK inhibitor U0126 or the Hsp70 selective inhibitor pifithrin‐μ prior to our behavioral assays, and found that ERK inhibition completely recapitulated the effects of Hsp90 inhibition on tail‐flick and paw incision anti‐nociception. Hsp70 inhibition had no effect, suggesting that it is not the mechanism for Hsp90 regulation of opioid anti‐nociception. Taken together, these findings suggest that Hsp90 promotes opioid‐induced anti‐nociception by facilitating ERK activation in mouse brain. These findings broaden our understanding of the role of Hsp90 in opioid signaling and behavior, and may suggest future therapies to improve the therapeutic index of opioid drugs, such as an Hsp90 activator.

Support or Funding Information

Institutional support from the Universities of Arizona and New England, and a Pilot Project Grant (JM Streicher, PI) under an institutional COBRE award (P20GM103643, I Meng, PI).
The Marine Pharmacology and Pharmaceuticals Pipeline in 2016
Authors: Mayer Alejandro M , Nguyen Michelle , Kalwajtys Patrycja , Kerns Hillary , Newman David J , Glaser Keith B
Published: November 2023

The pharmaceutical potential of the enormousbiodiversity of organisms present in the world's oceans continues to be the focus of intense global preclinical and clinical research. In October 2016 there were six marine‐derived drugs approved by the FDA: for cancer, cytarabine (Cytosar‐U®, Depocyt®, FDA‐approved 1969), eribulin mesylate (Halaven®, FDA‐approved 2010), brentuximabvedotin (Adcetris®, FDA‐approved 2011, and, trabectedin (Yondelis®, FDA‐approved2015); for pain, ziconotide (Prialt®, FDA‐approved 2004); forhypertriglyceridemia, omega‐3‐acid ethyl esters (Lovaza®, FDA‐approved 2004). The clinical marine pharmaceuticalpipeline in 2016 consisted of multiple compounds in clinical trials: 5 marine‐derived compounds in Phase III, 8 compounds in Phase II, and 14 compounds in Phase I, many of them auristatin‐containing antibody drug conjugates. Regular updates on the clinical marine pharmaceutical pipeline are posted at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Furthermore, the preclinical marine pharmacology pipeline continued to be very productive as highlighted by the over 500 manuscripts published during the past 21 months by MARINE DRUGS, a journal that publishes original research on the pharmacology of drugs from the sea: http://www.mdpi.com/journal/marinedrugs. We conclude that in late 2016, both the marine pharmacology preclinical and clinical pharmaceuticalpipelines remained very active.

Support or Funding Information

Supported by Midwestern University.