Podcasts about n2a

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Latest podcast episodes about n2a

Riding Shotgun With Charlie
RSWC #214 News2A

Riding Shotgun With Charlie

Play Episode Listen Later Oct 23, 2024 44:34


Riding Shotgun With Charlie #214 News2A Shawn Abramson & Grant Kielczewski   Last year, I filmed a show with Shawn and Grant from News2A. Due to camera issues, we decided to scrap it and do it another time. That time was in Knoxville, Tenn, at the Gun Owners of America's G.O.A.L.s event. Since we first tried this, Grant up and moved from New Jersey to middle Tennessee, so this was the ideal time to make it happen again.    Shawn grew up in a mostly non-gun house, but he and his brother took any chance they could to go out and shoot with their father's friends. At 18, getting an FID and joining the NRA was on the top of the things he wanted to do as an adult. For Grant growing up in the Midwest, guns were just tools. Also buying his first gun at 18, he spent years just being a casual shooter. In the last five years, things changed for him. He's been more active in taking courses and in helping others get started on their gun owning path. He's been working more towards becoming a better instructor.    Shawn and Grant were both New Jersey guys with an idea to start a website with a focus on the Garden State.  News2A (N2A) was launched after the Bruen decision in June 2022. New Jersey finally caved, kicking and screaming, to allow their peasants to have pitchforks. And allow them to carry their pitchforks. For a while, it was a hotbed of overreaching government having a post-Bruen hissy fit. There were lots of things going on. The gents thought there needed to be a place where people could get the up to date and correct info on the goings on.   We spend some time talking about the differences between states like, New Jersey & Massachusetts, versus other states, like Florida & Tennessee. It is bad in Jersey. You cannot carry in sensitive places so you have to disarm in your car and do a lot of administrative loading and unloading. Post Bruen, New York and New Jersey really tried tamping down the rights and abilities of gun owners.    When the guys started getting more involved, they went to Trenton to watch the testimonies and see how the sausage is made. And it ain't pretty. None of the things we may have been taught in school, or from Schoolhouse Rock, really happens like that. It was an education and eye opening for them.    At one point, John Petrolino, RSWC #093, reached out to me and asked if I wanted to do some writing. He said that N2A was looking for writers and contributors for their website. He said I could send things to him to edit, then I could pass it along to N2A. In the meantime, he wrote to Shawn and Grant, saying that maybe they could publish some of my writings there. It's been a great outlet for me as a writer. I was able to cover the nonsense of the Gun Law Listening Tour in early 2023.    The website is chock full of writers. Covering topics of politics, gun rights, training, editorials. They've got about eight writers plus Shawn and Grant contributing to the site. Some I've had on the show and some still need to get in the stagecoach. They also have sections on the site that are great resources for Jersey residents, where you can carry, how to carry, how to apply for everyone. And, of course, safety tips, too.    Let me tell you, the gun folks from Jersey have aggravation in their blood about their process, licensing scheme, and the nonsense they have to deal with. There's only a handful of states that the Bruen decision really affected. These states are the most egregious about doing their best to make sure that lawful and responsible citizens have a hard time with owning, possessing, and carrying firearms. Fortunately, they've got Shawn and Grant putting in time, love, and money in keeping everyone informed and on top of the situation.  Favorite quotes: Shawn: “You meet the right people, too. They have the right values. They're just friendly people willing to help others in general.” Shawn: “They only care about making sure their party's agenda happens.” Grant:”We've tried to take this very complex information and make it as applicable to people as possible.” Grant: “I know people underestimate the value of training.” News2A Website https://www.news2a.com/   News2A Twitter/X https://twitter.com/News2ATeam   News2A Facebook https://www.facebook.com/news2a/   News2A Gab https://gab.com/News2A   US Law Shield (Code:NEWS2A) https://www.uslawshield.com/?affid=ad008c31-331d-11ef-bda8-0615552639c3 Second Amendment Foundation https://secure.anedot.com/saf/donate?sc=RidingShotgun    Citizens Committee for the Right to Keep and Bear Arms https://www.ccrkba.org/     Please support the Riding Shotgun With Charlie sponsors and supporters.    Dennis McCurdy Author, Speaker, Firewalker http://www.find-away.com/   Self Defense Radio Network http://sdrn.us/   Buy a Powertac Flashlight, use RSWC as the discount code and save 15% www.powertac.com/RSWC   SABRE Red Pepper Spray  https://lddy.no/1iq1n   Or listen on: iTunes/Apple podcasts https://podcasts.apple.com/us/podcast/riding-shotgun-with-charlie/id1275691565

PaperPlayer biorxiv cell biology
Alpha Synuclein Modulates Mitochondrial Ca2+ Uptake from ER During Cell Stimulation and Under Stress Conditions

PaperPlayer biorxiv cell biology

Play Episode Listen Later Apr 24, 2023


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.537965v1?rss=1 Authors: Ramezani, M., Wagenknecht-Wiesner, A., Wang, T., Holowka, D. A., Eliezer, D., Baird, B. Abstract: Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson' disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv neuroscience
Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Nov 10, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.09.514853v1?rss=1 Authors: Zhang, J., Zhang, M., Wang, Q., Wen, H., Liu, Z., Wang, F., Wang, Y., Yao, F., Song, N., Kou, Z., Li, Y., Guo, F., ZHU, S. Abstract: N-Methyl-D-Aspartate (NMDA) receptors are essential for many brain functions. These receptors are heterotetramers typically comprising two GluN1 subunits and two GluN2 subunits. The latter could alternate among four subtypes (N2A-N2D) and determine the functional diversity of NMDA receptors. For example, receptors containing N2C or N2D exhibit 50-fold lower channel open probability (Po) than those containing N2A. Structures of N2A- and N2B-containing receptors have been extensively characterized, providing molecular basis for understanding NMDA receptor function. Here we report the cryo-EM structures of N1-N2D and N1-N2C di-heterotetramers (di-receptors), and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a resolution up to 3.0 angstroms. Structural analysis showed that the bilobate N-terminal domain (NTD) in N2D adopted an intrinsic closed conformation, leading to a compact NTD tetramer in N1-N2D receptor. Functional studies further demonstrated that, in di-receptors containing N2D but not N2A or N2B, crosslinking NTD at the tetrameric interface had no effect on channel activity, while crosslinking ligand-binding domain (LBD) of two N1 protomers significantly elevated Po. Surprisingly, we found that the N1-N2C di-receptors spontaneously oscillated between symmetric and asymmetric conformation. The later one occupied a predominant population, whereby two N2C protomers exhibited distinct conformation. This asymmetry, which was also found to a lesser extent in N1-N2A di-receptor, was further locked by the binding of an N2C-specific allosteric potentiator PYD-106 to a unique binding pocket between NTD and LBD in only one N2C protomer. Finally, N2A and N2C in the N1-N2A-N2C tri-receptor displayed the conformation close to that found in one protomer of N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse functional properties of major NMDA receptor subtypes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv neuroscience
Synaptojanin1 regulates lysosomal functions in ventral midbrain neurons

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Oct 14, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512269v1?rss=1 Authors: Zhu, X., Prakash, S. S., McAuliffe, G., Pan, P.-Y. Abstract: A major pathological hallmark of Parkinsons Disease (PD) is the manifestation of Lewy bodies comprised of alpha-synuclein (a-syn). The accumulation of a-syn enriched protein aggregates is thought to arise from dysfunction in degradation systems within the brain. Recently, missense mutations of SYNJ1 encoding the SAC1 and 5-phosphatase domains have been found in families with hereditary early-onset Parkinsonism. Previous studies showed that Synj1 haploinsufficiency (Synj1+/-) leads to PD-like behavioral and pathological changes in mice, including the accumulation of the autophagy substrate p62 and pathological a-syn proteins in the midbrain (MB) and striatum. In this study, we aim to investigate the neuronal degradation pathway using the Synj1+/- MB culture as a model. Our data suggests that autophagy flux and cumulative autophagosome formation is unaltered at baseline in Synj1+/- MB neurons. However, lysosome number is reduced with a similar decrease in lysosomal proteins, including LAMP1, LAMP2, and LAMP2A. Lysosomes are hyperacidified with enhanced enzymatic activity in Synj1+/- MB neurons. Using a combination of light and electron microscopy, we show that lysosomal changes are primarily associated with a lack of SAC1 activity. Consistently, expressing the SYNJ1 R258Q mutant in N2a cells reduces the lysosome number. Interestingly, the lysosomal defects in Synj1+/- neurons does not impact the clearance of exogenously expressed wild-type a-syn; however, the clearance of a-syn A53T was impaired in the axons of Synj1+/- MB neurons. Taken together, our results suggest axonal vulnerability to lysosomal defects in Synj1 deficient MB neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

PaperPlayer biorxiv neuroscience
Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Sep 28, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.27.509765v1?rss=1 Authors: Sunna, S., Bowen, C., Zeng, H., Rayaprolu, S., Kumar, P., Bagchi, P., Guo, Q., Duong, D. M., Bitarafan, S., Natu, A., Wood, L., Seyfried, N. T., Rangaraju, S. Abstract: Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain are incompatible with the survival of adult murine neurons and contamination from non-target cell-types and experimental artifacts confound the data. To overcome these barriers, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes. LPS treatment induced greater than 500 differentially expressed proteins in transduced BV2 proteomes, including an increase in canonical M1 proteins (IL1a, Irg1, Oasl1) and a decrease in canonical M2 proteins (Arg1, MGl2). Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

PaperPlayer biorxiv neuroscience
Quercetin reduces APP expression, oxidative stress and mitochondrial dysfunction in the N2a/APPswe cells via ERK1/2 and AKT pathways

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Sep 19, 2022


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.18.508406v1?rss=1 Authors: Tang, Z., Guo, M., Peng, Y., Xiao, Y., Zhang, T., Ni, R., Qi, X. Abstract: Abnormal amyloid-{beta} (A{beta}) abnormal accumulation and oxidative stress play important roles in Alzheimer' disease (AD). Quercetin has been reported to possess antioxidant and anti-inflammatory properties, and thus of therapeutic interests for neurodegenerative disorders. In the present study, we aimed to characterize the mechanisms by which quercetin exerts neuroprotective effects in murine neuroblastoma N2a cells stably expressing human Swedishh mutant amyloid precursor protein (APP). Quercetin treatment exhibited low cytoxicity, attenuated APP expression and APP-induced oxidative neurotoxicity in N2a/APP cells. We found that quercetin effected via the down-regulation of phospho-extracellular signal regulated protein kinase (p-ERK1/2) pathway and up-regulation of phospho-protein kinase B (p-AKT) pathway in N2a/APP cells. In addition, quercetin ameliorated the elevated levels of reactive oxygen species using DCFH-DA flow-cytometry in N2a/APP cells, lipid peroxidation using (4-HNE), and DNA oxidation (8-OHdG assays). Quercetin ameliorated the loss of mitochondrial membrane potential using JC-1 fluorescence assay in N2a/APP cells in a dose-dependent mannor. In conclusion, we domenstrated the neuroprotective effects of quercetin against the APP expression induced oxidative neurotoxicity, impairment of mitochondrial function and oxidative stress through inactivation of the ERK1/2 signaling pathway and activation of AKT signaling pathways. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Levantine Arabic, made easier
Episode 36: Girls on Wheels

Levantine Arabic, made easier

Play Episode Listen Later May 11, 2021 19:49


Founder of Girls on Wheels Lina Al Kassem is Uniting & empowering women on rollers, skateboards, bicycles & motorbikes. Listen to this wonderful episode & support their mission by buying a pair of socks!  Check out Lina's work below:  https://www.instagram.com/girlsonwheelslb/ https://www.instagram.com/girlsonwheelsuae/ Make sure to check out our Top 20 Vocabulary picks from this episode below.  Join our Conversational Classes:  https://nasmaofny.com/adult-group-conversation-class/ Join our Levantine Masterclass: https://nasmaofny.com/online-course-membership/ Get the eBook: https://nasmaofny.com/adult-textbooks/ Follow us on Instagram: https://www.instagram.com/levantinearabic_bynasmaofny/ Subscribe to our Youtube channel: https://www.youtube.com/channel/UCZ3xodgJWwMeRCE59ZNNCTQ?view_as=subscriber Music by: Stay Young by Nick Petrov Vocabulary: 1. Nade/ نادي: Gym or club 2. Mashro3/ مشروع: Project  3. Tkaser 7alak/ حالك تكسر: To break  4. Khalfiyeh Riyadiyeh/ خلفية رياضية: Sports background or physical fitness background  5. Menfeyeh/ منفية: Isolated  6. Nashatat/ نشاطات: Activities  7. Nasher/ نشر: Post 8. Nsh-je3/ نشجع: Support  9. Ted3amo/ تدعمو: You support  10. Bighat el-nazar/ بغت النظر: Regardless  11. Kalset/ كلسات: Socks  12. Drus majeneyeh/ دروس مجانية: Free Lessons  13. Dar-reb/ درب: Coaching  14. 3er2et ejro/ عرقت اجرو: His leg was sweating  15. Bi7efele/ بحفلي: Rubs  16. N2a-le3/ نقلع: To flourish  17. Estedaftek/ استضافتك: For your feature  18. Dawe/ ضوي: To shine or light on 19. Bitmeres/ بتمارس: I practice 20. Titwasal/ تتواصل: Communicate 

PaperPlayer biorxiv neuroscience
Optineurin promotes aggregation of mutant huntingtin and mutant ataxin-3, and reduces cytotoxicity of aggregates

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Aug 13, 2020


Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249201v1?rss=1 Authors: Moharir, S. C., Raghawan, A. K., Swarup, G. Abstract: Optineurin (OPTN), a cytoplasmic adaptor protein involved in cargo selective autophagy of bacteria, damaged mitochondria and mutant protein aggregates, is frequently seen in pathological structures containing protein aggregates, associated with several neurodegenerative diseases. However, the function of OPTN in these protein aggregates is not known. Here, we have explored the role of OPTN in mutant protein aggregation and in cytoprotection from toxicity of mutant proteins. Mutant huntingtin (mHtt) and mutant ataxin-3 (mAtax-3) showed reduced formation of aggregates in Optn-/- mouse embryonic fibroblasts as compared with wild type cells. Co-expression of OPTN enhanced aggregate formation by mHtt and mAtax-3 in Optn-/- cells. C-terminal domain of OPTN (412-577 amino acids) was necessary and sufficient to promote aggregate formation by these mutant proteins. The E478G mutant of OPTN, defective in ubiquitin-binding and autophagy, was also able to promote aggregation of mHtt and mAtax-3. OPTN and its C-terminal domain form a complex with the chaperone HSP70 known to promote mutant protein aggregation. Overexpression of mHtt or mAtax-3 induced more cell death in Optn-/- cells compared with wild type cells. Importantly, compared to wild type cells, Optn-deficient cells having mHtt or mAtax-3 aggregates showed higher level of cell death in neuronal (N2A) and non-neuronal cells. Our results show that OPTN promotes formation of mutant huntingtin and mutant ataxin-3 aggregates, and this function of OPTN might be mediated through interaction with HSP70 chaperones. Our results also show that OPTN reduces cytotoxicity caused by these mutant protein aggregates. Copy rights belong to original authors. Visit the link for more info

Der Morlock Gruppe
Folge 20: Spiral

Der Morlock Gruppe

Play Episode Listen Later May 5, 2019 84:09


In der heutigen Morlock Gruppe geht’s um die neue Fraktion Spiral, die zwar sehr viele Einheiten von Tohaa nutzt, aber kein Tohaa-Sektor ist, sondern zu N2A verordnet wurde. Nun, die Wege von Corvus Belli sind unergründlich und so fragen wir da auch nicht weiter nach. Da aber Tohaa und Spiral thematisch sehr eng zusammenhängen, ist diese Folge eigentlich eher der erste Teil der Armeevorstellung von Tohaa. Da ich selbst - wie könnte es auch anders sein - keine Ahnung von Spiral oder von Tohaa habe, habe ich mir ein wenig Verstärkung gesucht. Vorgesehen waren eigentlich Fabian und Jonas. Allerdings ist Fabian ein wichtiger Termin dazwischen gekommen, sodass mir Jonas als einziger Gesprächspartner blieb. Das macht aber gar nichts, da Fabian für uns noch die News eingesprochen hat. So oder so wünschen wir euch viel Spaß mit der Episode. Ein kleiner rechtlicher Hinweis: Für das Intro & Outro wurde der Titel "Future Buzz" von Tim van der Beek verwendet, der einer CC-BY-4.0-Lizenz unterliegt. (https://creativecommons.org/licenses/by/4.0/deed.en_GB) Für das News-Intro wurde „News Intro“ von Maximilien verwendet, der einer CC-BY-3.0-Lizenz unterliegt. (https://creativecommons.org/licenses/by/3.0/)

Cars Yeah with Mark Greene
538: Gene Langmesser is the President and CEO of N2A Motors in Orange County California.

Cars Yeah with Mark Greene

Play Episode Listen Later Jun 23, 2016 36:43


Gene Langmesser is the President and CEO of N2A Motors in Orange County California. N2A Motors is an auto manufacturer with a mission of reviving the art of American coach building. The N2A moniker is an abbreviation of “No Two Alike” which profiles the company vision. He and his talented team take everything you love about automobiles, classics, retro, and modern sports car and they build them in to new vehicles with state-of-the-art performance, safety, and reliability. Gene’s extensive years of domestic and international experience in both aerospace and automotive sectors bring N2A Motors viable leadership.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 11/19
Fluorescence labeled PEI-based gene delivery systems for near infrared imaging in nude mice

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 11/19

Play Episode Listen Later Mar 4, 2010


Gene therapy is a research area where nucleic acids are transferred into cells to treat neoplastic, metabolic and hereditary diseases. Delivery of genetic material into living organisms can be achieved with viral or non-viral vectors. Viral gene carriers are very efficient but present some major disadvantages due to their pathogenicity and immunogenicity. Nonviral carriers are based on synthetic molecules binding and condensing nucleic acids into small, virus-like particles. The aim of this thesis was to study the biodistribution and tumor targeting properties of non-viral gene vectors based on polyethylenimine (PEI) after systemic injection into mice. The gene vectors were labeled with fluorescent dyes emitting in the near infrared (NIR), which allowed studying their bio-distribution in living animal over time. Owing to its amine groups PEI has a high positive charge density that enables electrostatic interactions with negatively charged nucleic acids and their efficient compaction into nucleic acid-PEI complexes, called polyplexes. The net positive surface charge of these polyplexes permits interactions with negatively charged cell surface molecules, thus leading to their internalization into the cell. To avoid unspecific interactions with blood components and nontarget tissues after intravenous application, polyplexes were shielded with the hydrophilic molecule polyethylene glycol (PEG). PEI-based gene carrier systems were tested on two subcutaneously implanted tumor types: Human Hepatocellular Carcinoma (HUH7) and Murine Neuroblastoma (N2a). HUH7 cells express epidermal growth factor (EGF) receptors, while N2a cells express transferrin (Tf) receptors on their surfaces. To enable targeting of the polyplexes to the tumor cells, polyplexes were generated containing the ligands EGF and Tf for targeting of HUH7 cells and N2a cells respectively. The targeted polyplexes were then intravenously injected into immunodeficient, athymic nu/nu mice in which HUH7 or N2a tumor cells had been previously set under their skin. To monitor the biodistribution of polyplexes throughout the mouse organism and to evaluate their gene delivery capability into the neoplastic cells, polyplexes were labeled with fluorescent dyes (Alexa 750, NIR 797) or near infrared emitting quantum dots (QD), whose fluorescent expression signal was detected and analyzed with a device for imaging in vivo. All fluorescent molecules and quantum dots were biocompatible and non-toxic. They emitted light in the near infrared area of the spectrum, thus avoiding overlapping phenomena with autofluorescent biomolecules or absorption of light by hemoglobin. With all dyes used for polyplex labeling a fluorescent signal could be observed in organs like liver and lung being clearly distinguishable from background fluorescence. Among the fluorescent molecules tested, quantum dots were identified being the most suitable method for in vivo studies, showing the highest signal/noise ratios. PEG-shielding led to best tumor targeting efficiency when administering EGF or Tf-targeted polypelxes in mice bearing HUH7 and N2a tumors respectively. A clear fluorescent signal specific for tumor tissue was detected; the imaging software used allowed quantitative analysis of this signal. For this reason this system is now available for further experimental applications.

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06
Characterization of the 37-kDa/67-kDa laminin receptor as the cell surface receptor for the cellular prion protein

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Mar 25, 2002


Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPSc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies (TSEs), which affect both, humans and animals. Human prion diseases occur in infectious, sporadic or genetic forms. The "protein only" hypothesis argues that the key event in the pathogenesis represents the conversion of the normal host protein, PrPc, into its pathogenic isoform PrPSc. Prion diseases have been associated with the accumulation of this abnormally folded protein and its neurotoxic effects. However, it is not known if PrPc loss of function is an important factor since the normal biological function of PrPc, a cell surface-anchored glycoprotein predominantly expressed in neuronal cells, and the cellular processes in which this protein is involved remain obscure. Recently, the human 37 kDa laminin receptor precursor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), was identified as a binding partner for the cellular prion protein in a yeast two-hybrid screen. In order to characterize the possible role of LRP/LR as a cell surface receptor for PrPc, cell culture studies were performed to investigate the cellular localization of PrP and LRP/LR and to analyse the binding and internalization behaviour of PrP depending on the presence of LRP/LR on the cell surface of neuronal and non-neuronal cells. Immunofluorescence analysis of non-permeabilized murine neuroblastoma cells demonstrated that PrP and LRP/LR co-localize on the surface of these cells. In addition, baby hamster kidney (BHK) cells transfected with recombinant Semlik-Forest virus RNAs overexpressed human PrP and human LRP at their cell surface, the latter one orientated as a type II transmembrane protein with its C-terminus outside and its N-terminus inside the cell. Co-localization of both proteins was observed on BHK cells co-transfected with LRP and PrP encoding recombinant SFV RNAs. Cell binding and internalization assays with recombinant human PrP demonstrated the LRP/LR-dependent binding and endocytosis of externally added human PrP. An increased, dose-dependent cell binding of recombinant PrP was demonstrated by BHK cells overexpressing full-length human LRP on their cell surface. Trypsin treatment of the cell surface revealed the LRP dependent internalization of GST-tagged and untagged, glycosylated PrP. In contrast to wild-type LRP, the expression of an LRP mutant lacking its transmembrane domain led to the secretion of this mutant from transfected BHK cells and totally abolished the binding and internalization of exogenous, recombinant PrP. This LRP mutant could function as a decoy recetor in therapy of TSEs. The strict LRP/LR specificity of the PrP binding to neuronal cells was verified by testing the displacement capacity of a series of different antibodies in the LRP-PrP binding reaction. Only LRP and PrP specific antibodies were able to block totally the binding of human GST-fused PrP to N2a and NT2 cells whereas various control antibodies used for competition showed no effect. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of cellular PrP to the 37-kDa/67-kDa LRP/LR. The relationship between the 37-kDa LRP and the 67-kDa high-affinity LR is unknown so far. Both forms were observed in plasma membrane fractions of N2a cells. We conclude from these data that the 37-kDa/67-kDa laminin receptor acts as the main cell surface receptor for PrP. High-level expression and purification of recombinant, glycosylated prion proteins in mammalian cells is essential for a better understanding of the physiological function of PrPc and biochemical processes responsible for prion diseases. Due to the presence of important organelles, membranes and other cellular cofactors which are necessary for the correct processing, trafficking and localization of prion proteins mammalian cell culture systems such as the Semliki-Forest virus (SFV) system allow the synthesis and characterization of wild-type as well as mutant PrP to get a better insight into the biology of these proteins. Therefore, the SFV system was used to generate recombinant highly glycosylated human wild-type and human disease-associated mutant prion proteins as well as FLAG-tagged human and bovine PrP in cultured BHK cells. Both mutated variants, which are related to the human prion diseases fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) reveal proteinase K (PK) resistance, one of the most typical biochemical properties characteristic for the infectious scrapie isoform of the prion protein. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP without any significant differences regarding the cellular distribution and expression level. In addition, FLAG-tagged prion proteins were expressed with high efficiency in BHK cells showing the typical glycosylation pattern allowing the rapid and simple purification via anti-FLAG antibody chromatography. PrP dimers could play an essential role in the PrPc to PrPSc conversion process and might be involved in PrP interspecies transmission. Recently, crystallization of the prion protein in a dimeric form was reported. Size exclusion chromatography showed that native soluble homogeneous FLAG tagged prion proteins from hamster, man and cattle expressed in the baculovirus system were predominantly dimeric. The PrP/PrP interaction was confirmed in rec. SFV-RNA transfected BHK cells co-expressing FLAG and oligohistidine tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. The identification of the 37-kDa/67-kDa laminin receptor as the receptor for the cellular prion protein might represent an important step for a better understanding of the molecular biology of prion diseases and might lead to the development of powerful therapeutics such as LRP/LR specific antibodies for the treatment of these unconventional diseases.