bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 Information2T32NS00748016 (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 InformationBranfman 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 InformationThis 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 InformationThe 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 InformationThis 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 InformationInstitutional 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 InformationSupported by Midwestern University.
Development of a Novel Single‐Cell Assay to Interrogate a CRISPR/Cas9 Library for Regulators of MEK Kinase 2
Authors: Lotesto Krista , Cuevas Bruce 
Published: November 2023

MEK Kinase 2 (MEKK2) controls activity of the mitogen‐activated protein kinase ERK5 and in vivo models strongly suggest that MEKK2 activity is an important contributing factor in diseases ranging from cardiomyopathy to cancer. Thus, MEKK2 and the signaling proteins that control MEKK2 activity represent potential therapeutic targets. However, pharmacological MEKK2 inhibitors are not currently available and MEKK2 regulation is poorly understood. CRISPR/Cas9‐based genetic screening represents a powerful technology for defining regulatory networks at the single‐cell level, but single‐cell assays of MEKK2 required for CRISPR/Cas9 screening have not been described. Herein, we report our development of a Fluorescence Activated Cell Sorting (FACS)‐based MEKK2 signaling assay that can be used to screen a CRISPR library to identify MEKK2 regulators. We examined the utility of MEKK2‐dependent ERK5 phosphorylation as a surrogate indicator of MEKK2 activity in several human cell lines by quantifying ERK5 activation loop phosphorylation (pERK5) with indirect immunofluorescence. We determined the pERK5 fluorescence signal intensity induced by multiple reported ERK5‐activating stimuli in multiple human cell lines, and performed z′ factor analysis to establish the preferred conditions for a FACS‐based separation of cells with active MEKK2 from cells with inactive MEKK2. We discovered that anti‐pERK5 staining of 293T cells stimulated with MEKK2 activator pervanadate provides a means to effectively separate cells with activated MEKK2 from non‐treated cells, and allows us to distinguish and separate the two populations by FACS. Our data suggest that fluorochrome‐linked anti‐pERK5 can be used to sort cells that express CRISPR gRNA that inhibit MEKK2 activity, thereby identifying MEKK2 regulators. Therefore, we conclude that anti‐pERK5‐based immunofluorescence coupled with FACS analysis provides a new tool for performing unbiased genetic screens utilizing CRISPR/Cas9 library‐expressing cells to identify MEKK2 regulators in order to identify novel pharmacological targets for the treatment of human disease.Support or Funding InformationASPET “Discover Pharmacology” Summer Undergraduate Research Fellowship