Podcasts about agrin

What a better way to start the new 2024 year than to learn about the current, realistic prospect of living longer and better lives due to the latest advances in medicine and pharmaceutical research from a preventive and longevity medicine advocate, researcher, and expert, James Peyer, PhD. This episode features a personal, candid story of his journey from academia into the industry, inspiration behind pursuing preventive healthcare and longevity treatments, and latest new treatments in human trials that increase healthspan and lifespan from a keynote speech at the 2nd annual global Medical Affairs Innovation Olympics Opening Ceremony in 2023. Subscribe to Alloutcoach and share this episode with your friends! 0:00 Episode Introduction 5:10 James Peyer's Keynote Speech Outline 7:14 James Peyer's late grandfather and inspiration to pursue prevention medicine 7:55 Frustration with Health System's Interaction with Elderly 9:53 Early animal experiments in genes doubling lifespan 11:14 80+ treatments that extend healthy lifespan in animal models today 13:09 Real-world reason why human trials in slowing aging did not advance 14:10 Breakthrough Idea to Advance Prevention Medicine in Humans 16:35 Cambrian Bio's unique Longevity Platform Ecosystem 19:42 Two Longevity Medicine Biotech Case Studies 20:50 Amplifier Therapeutics - a Cambrian Bio Pipeline Company 23:48 New Promise in 10-year race to develop an AMPK (central energy regulator) activator 25:40 ATX-304: new AMPK candidate in human trials 27:50 Second Vignette: Velakor - agrin-induced pleiotropic cardiac repair 31:11 Velakor's Agrin in human clincal trials in 2024 to regenerate heart muscle 33:25 Q1: Regulatory approval considerations for other longevity medicine companies 34:08 Conduct Primary and Secondary Prevention Trials - FDA supportive of preventive treatments 35:21 FDA interested in preventive medicine, transition from sick care to preventive care in US 37:11 Q2: Difference between healthspan and lifespan, and impact on drug development 38:22 Patient Survey Data on Critical Value of Quality not only Length of Life 41:25 Q3: Technological innovations most promising in increasing longevity and healthspan 43:49 James' Final Message on Innovation and Entrepreneurship

Why did people take to the streets, risking arrest and a barrage of bullets? After protests turned violent and hundreds of people were killed, four Iranians tell the story of why they risked their lives. What has been happening in Iran to drive them out onto the streets to face bullets? ‘Agrin' tells Phoebe Keane she's tired of being objectified as a woman, and having no faith that the authorities will take sexual assault seriously when the police themselves are accused of raping prisoners. Mahsoud tells how he was shot during a protest but feared going to the hospital in case the authorities put him in jail. When plain clothed police loitered outside his family home, he decided to leave Iran. Still bleeding and with a metal pellet lodged in his ear impairing his hearing, he finally made it across the border to Iraq. ‘Nazy' tells of being arrested by the morality police while walking to work and being shoved in a van as the heels on her shoes were too high. She started to protest every day and now walks through the streets with her hair blowing in the wind, an act of defiance. ‘Farah' remembers a time in Iran when women could dance and sing in public and protests because she wants her daughter to live a life without fear. Presenter: Phoebe Keane Producers: Ed Butler, Ali Hamedani, Khosro Isfahani and Taraneh Stone Series editor: Penny Murphy

Why did people take to the streets, risking arrest and a barrage of bullets? After protests turned violent and hundreds of people were killed, four Iranians tell the story of why they risked their lives. What has been happening in Iran to drive them out onto the streets to face bullets? ‘Agrin' tells Phoebe Keane she's tired of being objectified as a woman, and having no faith that the authorities will take sexual assault seriously when the police themselves are accused of raping prisoners. Mahsoud tells how he was shot during a protest but feared going to the hospital in case the authorities put him in jail. When plain clothed police loitered outside his family home, he decided to leave Iran. Still bleeding and with a metal pellet lodged in his ear impairing his hearing, he finally made it across the border to Iraq. ‘Nazy' tells of being arrested by the morality police while walking to work and being shoved in a van as the heels on her shoes were too high. She started to protest every day and now walks through the streets with her hair blowing in the wind, an act of defiance. ‘Farah' remembers a time in Iran when women could dance and sing in public and protests because she wants her daughter to live a life without fear. Presenter: Phoebe Keane Producers: Ed Butler, Ali Hamedani, Khosro Isfahani and Taraneh Stone Series editor: Penny Murphy

Dr. Elahi Behzad interviews Dr. Michael H. Rivner on the clinical features of LRP4/agrin-antibody–positive Myasthenia Gravis.

Dr. Elahi Behzad interviews Dr. Michael H. Rivner on the clinical features of LRP4/agrin-antibody positive Myasthenia Gravis (MG).

This week’s episode includes author Allan Sniderman and Associate Editor Anand Rohatgi as they discuss the expected 30-year benefits of early versus delayed primary prevention of cardiovascular disease by lipid lowering and management. TRANSCRIPT: Dr Greg Hundley: Welcome everyone to this September 1 issue, as we start into the fall in North America, and I guess we're getting into spring-ish in the Southern hemisphere. Today, it's just myself, Dr Greg Hundley, Associate Editor and Director of the Pauley Heart Center at VCU Health in Richmond, Virginia. And, I'm so sad because my good friend, Carolyn, cannot be with us today. However, we have a great feature for the latter half of this recording and discussing some of the benefits of primary prevention using lipid lowering therapy to help prevent cardiovascular disease. But, before we get to that, let's grab a cup of coffee and let's go through some of the other articles in this issue. So, the first one is from the world of basic science and it's from Professor Eldad Tzahor, from the Weizmann Institute of Science. And, it's focusing on Agrin. So, this team previously reported that a fragment of the extracellular matrix protein, Agrin, promoted cardiac regeneration following myocardial infarction in adult mice. And, in this study the investigators propose to test the therapeutic potential of Agrin in a preclinical porcine model. They performed ischemia reperfusion injuries using balloon occlusion for 60 minutes, followed by either a 3, 7, or 28-day reperfusion period. They demonstrated that local antegrade delivery of recombinant human Agrin, or RH Agrin, to the infarcted pig heart can target the effected regions in an efficient and clinically relevant manner. In fact, a single dose of recombinant human Agrin improved heart function, reduced infarct size, reduced fibrosis and reduced adverse remodeling parameters, 28 days post myocardial infarction. Short-term myocardial infarction experiments, along with complementary mirroring studies, revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as aggregation mechanisms of action. So in summary, this team demonstrated that a single dose of Agrin was capable of reducing ischemia reperfusion injury and improving heart function. Demonstrating that Agrin could serve as a therapy for patients with acute MI and potentially heart failure. So, this set the stage for future studies in human subjects. Okay. Well, our next paper is clinical and evaluates exposure to air pollution and particle radioactivity with a risk of ventricular arrhythmias. And, it comes to us from Ms. Ajani Peralta from Harvard University. Now, individuals are exposed to air pollution and ionizing radiation from natural sources through inhalation of particles. So, in this study, the team investigated the association between cardiac arrhythmias and short-term exposures to find particulate matter, PM 2.5, and particle radioactivity. So, ventricular arrhythmogenic events were identified among 176 patients with dual chamber implanted cardio defibrillators in Boston, Massachusetts, between the period of time of September 2006 and June 2010. And, patients were assigned exposures based on their residential addresses. So, what did they find? Well, in this high-risk population, those with these defibrillators, intermediate, 21-day parts per million, 2.5 exposure was associated with higher odds of a ventricular arrhythmia event onset among those with known cardiac disease and indication for ICD implantation. But this was independent of particle radioactivity. So, important information coming to us relating to air pollution and ionizing radiation in relation to ventricular events. Next, let's get back to another informative study from the world of basic science. And, this one involves genomic binding patterns of forkhead box protein 01 and how that is implicated in the development of cardiac hypertrophy. The study comes to us from Walter Koch from Temple University and their co-investigators. So, cardiac hypertrophic growth is mediated by changes in gene expression, as well as changes that underlie the increase in cardiomyocyte size. The former is regulated by ischemia reperfusion or loss, while the latter involves incremental increases in the transcriptional elongation activity of Pol II, that is preassembled at the transcription start site, or TSS. The differential regulation of these two distinct processes, by transcription factors, really hasn't been explored. So, this group sought to investigate the forkhead box protein, and we're going to call it FOX01, which is an insulin sensitive transcription factor that is regulated by hypertrophic stimuli in the heart. To date, however, the scope of its gene regulation is also somewhat uncertain. So, to address this, the investigators performed FOX01 chromatin immunoprecipitation deep sequencing, or ChIP-sequencing, in mouse hearts following seven-day isoproterenol injections, transverse aortic constriction, or vehicle injection by sham surgeries. The investigators found that FOX01 may mediate cardiac hypertrophic growth via regulation of Pol II de novo recruitment and pause release. As the latter represents the majority, or almost 59% of FOX01 bound Pol II regulated genes following pressure overload. So, in conclusion, these findings demonstrate the breadth of transcriptional regulation by FOX01 during cardiac hypertrophy, which is important information that should be valuable for future therapeutic targeting. Moving on from basic science and coming back into the world of clinical science. And, this next paper is from Professor Sripal Bangalore from New York University School of Medicine. And, it involves routine revascularization versus initial medical therapy in those with stable ischemic heart disease. So, coronary arterial revascularization is often performed, as we know, in patients with stable ischemic heart disease. And, these authors conducted a PubMed Embase central search for randomized trials, comparing routine revascularization versus an initial conservative strategy in patients with stable ischemic heart disease. The primary outcome was death, and secondary outcomes included cardiovascular death, myocardial infarction, heart failure, stroke, unstable angina, and freedom from angina. And, the trials were stratified by percent stent use, and by percent statin use, to evaluate the outcomes across all of these trials. So, 14 randomized clinical trials that enrolled 14,877 patients followed up for a weighted mean of 4.5 years with a total of 64,678 patient years of follow-up, were used for the study. Most of the trials enrolled patients with preserved left ventricular systolic function, low symptom burden, and they excluded patients with left main coronary artery disease. So, here are the results, revascularization compared with medical therapy alone, was not associated with a reduced risk of death. The trial sequential analysis showed that the cumulative Z curve crossed the futility boundary indicating firm evidence for lack of a 10% or greater reduction in death. Now, revascularization was associated with a reduced non-procedural MI rate, but also with an increased procedural MI rate with, therefore, no overall difference in myocardial infarction incidents. So, another point in this study is that there was a significant reduction in unstable angina and increase in freedom from angina was observed in those that underwent revascularization. Finally, there were no treatment related differences in the risk of heart failure or stroke. So in conclusion, in patients with stable ischemic heart disease, routine revascularization was not associated with improved survival, but was associated with a lower risk of non-procedural myocardial infarction, and unstable angina with greater freedom from angina at the expense of higher rates of peri procedural myocardial infarction. Now, longer term follow-up of trials is needed to assess whether the reduction in these non-fatal spontaneous events actually improves long-term survival. Well, listeners what else is in the issue? Now, we refer to this as what's in the mailbox? So, we have a research letter from Jianyi Zhang regarding the apical resection prolongs the cell cycle activity and promotes myocardial regeneration, after left ventricular injury, in the neonatal pit. We have another research letter from Roger Foo, assigning distal genomic enhancers to cardiac disease-causing genes. What else is in the issue? There's a nice, On My Mind piece, from Anthony Wierzbicki on phenomics, not genomics, for cardiovascular risk assessment. And, there's a Perspective piece from Dr Dana Gal, regarding considerations for triaging elective cases in children with cardiac disease in a time of crisis. Finally, we have an exchange of letters from Dr Daxin Wang and Dr Prabhakara Nagareddy regarding the previously published article, “Neutrophil Derived S100A8/A9 Amplification of Granulopoieses After Myocardial Infarction.” There's a very nice case series regarding an unusual reversible cause of acute high output heart failure complicated by refractory shock from Dr Matthew Durstenfeld. And then, finally, we have an ECG challenge from Mr. Alejandro Cruz-Utrilla, regarding giant T-wave inversion and dyspnea in the time of this coronavirus pandemic. Well, listeners, what a great issue. And now, we get to look forward to that feature discussion from Dr Allan Sniderman, regarding the benefits of early versus delayed primary prevention by lipid lowering therapy. Well, listeners, now we get to move to our feature discussion today and understand a little bit more about lipid management. And, with us, we have Dr Allan Sniderman from McGill University, and our own associate editor, Dr Anand Rohatgi from University of Texas Southwestern in Dallas. Allan, we're going to start with you. Can you give us a little bit of information pertaining to the background, or the hypothesis? Why did you want to perform this particular study? Dr Allan Sniderman: Let us start from where we are in cardiovascular prevention now, which is the risk model. All of the major guidelines, throughout the world, select candidates for statin prevention based on their risk of a cardiovascular event over the next 10 years. Well, that's been a very positive development, but we need to appreciate what the limitations are. Because, tenure risk is so heavily based on age, what it boils down to is that if you're 60 and over, and a male you're going to be eligible. An increasing proportion of women will be eligible as they're older. But, if you're younger, regardless of your other causal factors, you may well not be eligible. And, the net result of that is, almost 50% of all cardiovascular events occur before 60, 65. So, half of the events are occurring before prevention even kicks in. The second point is that you can't get be at risk until you have disease within your arterial tree. So, what we've done is, we're trying to prevent the disease that's already present. So, although it's been a wonderful step forward, we think it's time to start moving beyond that model, and trying to prevent disease itself, rather than preventing people who've already had disease. So, what we decided to do, which we're shifting focus here from risk to causes. Risk, after all, is a consequence of causes. And, when you look at the pathology of atherosclerosis, with a few exceptions such as FH, up until the age of 30, 35, you don't have the complex lesions that can actually cause clinical events. So, we said, "Okay, we'll start at that time point. And, we'll try and quantitate the benefit of earlier intervention, versus later intervention, at different starting points." So, we based our analysis on NHANES, and we identified the people within the NHANES cohort, who would not have been eligible for prevention, based on the current American guidelines. So, we had three age starting categories, 30 to 39, 40 to 49, and 50 to 59. And, we followed them out for 30 years. Our analysis has the same duration of the follow up, but the same final date. That has to be kept in mind because it limits the total benefit on the early starters. And, we further sub divided the groups based on non-HDL cholesterol, into those who had a level above 160, those who had an intermediate level, and those who had a low level because we'd previously shown that non-HDL identifies a high-risk group over 20 to 30 years. We decided we'd look at two different models of the likelihood of the drug preventing events. One is the standard estimates that you would get from the statin clinical trials. But, if you reduce LDL cholesterol by a milli mol of 40 milligrams percent of your language, you reduce risk by about 22%. And, that's our conservative model. Then we took a model that's more biological and based on Mendelian randomization because, as you know, the Mendelian randomization analysis, the benefit, the reduction and event rate per milligram, per deciliter, lower LDL cholesterol is two or three fold greater than in the statin clinical trials. So, Brian Friends had produced a formula in which you blend those two together, the statin estimate and the Mendelian randomization and it varies depending on how early you start. And, that was a more optimistic model, obviously. But the major point I emphasize is that both models showed that if you start early, you do better. Now, how surprising is that? But the benefit depended on the level of non-HDL cholesterol. So starting at age 30, 35 in somebody who has a normal or low non-HDL cholesterol, doesn't really gain you all that much. Starting with somebody who has a high level, different story. In each of the categories, the conservative model, if you did the full 30-year prevention, you get a third to a half reduction in events. And, with the more optimistic model, you get half to two thirds reduction. The closer you get to where you'd start with the guidelines, you're losing that benefit. Then you say, "Okay, well, how optimistic is the optimistic model?" And, all the optimistic model is saying, "Let's remember how we get disease." If we stop the formation of a new lesion, well, that's perfect prevention. Well, we're doing it, present is trying to stabilize existing lesions. The older we get, the more disease we have, the less new lesion we have, the less potential for the big game in prevention. So stopping new lesion formation, it seems to me is from clinical reasoning, a pretty biologically and clinically coherent way to formulate what your prevention strategy should be. What we think we contributed, what we hope we've contributed with this analysis is saying, "Let's take a real step back, let's look at what we're doing and let's see how can we make a Magnus step forward in prevention?" And, the way we think you can do it is to start moving away from exclusively a risk paradigm, because that made sense. You want to treat people who were at risk. We get away from this 10-year duration and start focusing more on the causes, because when we look at an individual, we can measure the causes. Well, we talked about risks, that's a much slipperier concept, because it's a group. Is everybody in the group the same risk? They're clearly not. So, to try and get this to a much more concrete level, that's what we tried to do. Dr Greg Hundley: Very nice, Allen. And so, Anand, why did you select pushing this paper forward? And, what do you think this means for patients with high cholesterols? They're in their 40s and 50s. Help expand on some of what Allan has, so elegantly, described for us. Anand Rohatgi: First of all, Allan, thank you so much for doing this work and sending this to circulation. We were really excited to see this come across our desks, because at the end of the day, this manuscript, this study by Allan Sniderman and others, has very important public health consequences. And, that's why we were really interested in what they found and what they had to say about this topic. And, really what it comes down to is, like Allan mentioned, the trials for statin just really never addressed what to do about risk in younger individuals and over a longer period of time. And so, it was really these genetic studies, the Mendelian randomization studies, that really strongly showed the cumulative exposure effect of higher cholesterol levels, over time, and the benefits of maintaining really low levels. And so, what I think this paper does is it translates those scientific studies, those genetic studies, into a public health concept that's easily digestible for physicians, for clinicians, for people, for patients and stakeholders about what does this all mean? How can we benefit and when? And, I think it's very clear, the earlier you start, the more benefit you get over the life course. And, honestly, the take home message to me, it seems like for most of adulthood, the being able to maintain low risk and low cholesterol levels is the best path forward and not waiting until you find something wrong. And so, we were very, very excited about seeing that message translated in such an easy manner and compelling manner by Allan's work. Dr Greg Hundley: Very nice, 40 to 49-year old with non-HDL cholesterols greater than 160 milligrams per deciliter, would be expected to reduce their average predicted 30-year risk by 17%. That's just amazing. Allan, very quickly, in a minute, what do you think is the next study that needs to be performed in this area? Dr Allan Sniderman: I don't think we're ever going to get the primary prevention trial that we want, because it's too many people, too early, and we already know too much. I think the thing that we really need to do is figure out how we're going to most accurately measure the cause within the individual. I'll put in a plug for FOV, because I think it is a more effective measure, even the non-HDL cholesterol. And, I think that when we commit people or when we invite people to take medications early in life, we ought to be doing it on the strongest ground possible. I want to say thank you to be review process at Circulation, because the final paper that came out is not the paper that went in. The reviewers made superb, tough criticisms of this paper and not of the calculations, but of the way we were expressing it. And, they forced us to rethink and reimagine the context, in which the final form of this paper appears. So, we have a big thank you to our reviewers. We have a thank you to the patients of the editorial staff at Circulation. And, I have a big thank you to my collaborators, because they're really the strength of the paper. Dr Greg Hundley: Very nice, well listeners, it's been another great week here at Circulation and another very important piece of information in this feature discussion regarding monitoring and evaluating non-HDL cholesterol in the earlier years, and really considering initiation of statin therapy, to prevent the atherosclerotic lesions from forming, not just stabilize them later in life afterwards. Thanks to Anand Rohatgi and also Allan Sniderman for bringing us this wonderful article. We hope you have a great week. This program is copyright the American Heart Association 2020.  

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Myasthenia gravis (MG) ist eine neuromuskuläre Erkrankung mit einer Inzidenz von etwa 20-30/1 000 000 Menschen. Die Erkrankung führt zu einer Störung der synaptischen Erregungsübertragung an der neuromuskulären Endplatte und damit zu einer zunehmenden Schwäche der Muskulatur. In etwa 80 bis 90 Prozent der Patienten mit Myasthenia gravis konnten Autoantikörper gegen den Azetylcholinrezeptor nachgewiesen werden, die für die Symptome verantwortlich sind. In den letzten Jahren wurden bei einigen Myasthenia gravis Patienten Autoantikörper gegen die muskelspezifische Rezeptor-Tyrosinkinase (MuSK) und LRP4 (lipoprotein receptor-related protein) nachgewiesen. Allerdings gibt es immer noch eine geringe Anzahl von Myasthenia gravis Patienten, bei denen noch kein für die Beschwerden ursächlicher Antikörper gefunden werden konnte. Agrin ist neben LRP4 und MuSK ein weiteres Protein an der neuromuskulären Endplatte, und ist gemeinsam mit den beiden anderen Proteinen für die Bildung und Aufrechterhaltung der für die Erregungsübertragung notwendigen Aggregation von Azetylcholinrezeptoren verantwortlich. Deshalb liegt die Vermutung nahe, dass auch Autoantikörper gegen Agrin zu einer Beeinträchtigung der neuromuskulären Übertragung führen könnten. Auf dieser Hypothese beruht die vorliegende Studie, in der Seren von 54 Patienten mit klinisch diagnostizierter Myasthenia gravis auf das Vorhandensein von anti-Agrin-Antikörpern untersucht wurden. 30 der 54 untersuchten Seren waren seronegativ für Antikörper gegen den Azetylcholinrezeptor und MuSK, 15 Proben hatten erhöhte Konzentrationen an Antikörpern gegen MuSK und 9 davon waren seropositiv für anti-Azetylcholinrezeptor-Antikörper. Als Kontrollgruppe fungierten die Seren von 16 gesunden Probanden. Anstelle von Volllängen-Agrin wurde in den Experimenten mini-Agrin verwendet – ein synthetisches Protein, welches etwa 50 Prozent der Sequenz von Agrin enthält. Der Nachweis der Autoantikörper erfolgte mittels zweier unterschiedlicher Methoden: einem ELISA mit gereinigtem mini-Agrin und immunhistochemischen Färbungen von mit humanem mini-Agrin transfizierten HEK293 Zellen. Durch den ELISA gelang außerdem die Bestimmung der Antikörperkonzentrationen der detektierten anti-Agrin-Antikörper. In fünf der 54 untersuchten Seren konnten im ELISA erhöhte Konzentrationen an anti-Agrin-Antikörpern nachgewiesen werden. Die Antikörperkonzentrationen lagen zwischen 0,04 und 0,12 nM. Zwei dieser fünf Seren färbten spezifisch mit mini-Agrin transfizierte Zellen an, drei davon zeigten eine spezifische Färbung der neuromuskulären Endplatten in Mausgewebe. In vier der anti-Agrin-Antikörper-positiven Seren waren zuvor Antikörper gegen MuSK nachgewiesen worden; eines der Seren war anti-Azetylcholinrezeptor-Antikörper-positiv und zwei Seren hatten erhöhte Konzentrationen an Antikörpern gegen LRP4. Die Ergebnisse der vorliegenden Studie belegen das Vorhandensein von Antikörpern gegen Agrin in Patienten mit klinisch diagnostizierter Myasthenia gravis und geben damit Hinweise auf Agrin als ein neues Antigen in der Pathogenese der Erkrankung. In allen fünf der anti-Agrin-Antikörper-positiven Seren waren zuvor Antikörper gegen MuSK, LRP4 oder den Acetylcholinrezeptor nachgewiesen worden. Dies weist auf eine hohe Koinzidenz von Antikörpern gegen unterschiedliche Proteine der neuromuskulären Endplatte in Patienten mit Myasthenia gravis hin. Insbesondere konnten in dieser Studie zum ersten Mal drei Autoantikörper gegen Proteine der neuromuskulären Endplatte in einem Serum nachgewiesen werden. Inwiefern anti-Agrin-Antikörper an der Entstehung der für die Erkrankung typischen Veränderungen der Muskulatur beteiligt sind, bleibt weiterhin unklar und könnte Gegenstand zukünftiger Studien sein.

Muscle spindles are complex stretch-sensitive mechanoreceptors that consist of 4-12 specialized muscle fibers. These intrafusal muscle fibers are innervated in the central (equatorial) region by an afferent sensory axon and in both peripheral (polar) regions by efferent γ-motoneurons. Until now little is known about muscle spindle development at the molecular level, especially about the development of cholinergic specializations. My study shows that nicotinic acetylcholine receptors (AChR) are concentrated at the γ-motoneuron endplate as well as in the equatorial region. Moreover, enzymes required for the synthesis and removal of acetylcholine, including choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as vesicular acetylcholine transporter (VAChT) and the AChR-associated protein rapsyn are all concentrated at the polar γ-motoneuron endplate and (with the exception of AChE) also at the equatorial region. Finally, the presynaptic protein bassoon, involved in synaptic vesicle exocytosis, is also present at the γ-motoneuron endplate and at the annulospiral sensory nerve ending. During postnatal development, the AChR subunit composition at the γ-motoneuron endplate changes from the γ-subunit containing fetal AChR to the ε-subunit containing adult AChR. This is similar to the postnatal change at the neuromuscular junction. In the equatorial region the ε-subunit expression starts around postnatal week two; however the γ-subunit persists in the central region despite the onset of the ε-subunit expression. Therefore, the γ- and ε-subunits are simultaneously present in the equatorial region. This result was confirmed using a mouse line in which the AChR γ-subunit was genetically labelled by green fluorescence protein (GFP). In this mouse, the GFP-labelled AChR γ-subunits are concentrated at the contact site of the intrafusal fiber with the sensory nerve ending. This result indicates different AChR maturation occurs within two areas of the same intrafusal fiber. I also show that agrin and the agrin receptor complex (consisting of LRP4 and MuSK) are present in muscle spindles in the region of the sensory and motor innervation. Moreover, agrin, MuSK, and LRP4 are expressed by proprioceptive neurons in dorsal root ganglia but only agrin and LRP4 were detected in the cell body of γ-motoneurons in the spinal cord. In mice with a targeted deletion of agrin, AChR aggregates are absent from the polar region and γ-motoneuron endplates do not form. By contrast, AChR aggregates remain detectable in the central part of intrafusal fibers. Moreover, muscle-specific re-expression of mini-agrin is sufficient to restore the formation of synaptic specializations at γ-motoneuron endplates. These results show an unusual AChR maturation at the annulospiral endings and confirm that agrin is a major determinant for the formation of γ-motoneuron endplates. Agrin on the other hand appears dispensable for the aggregation of AChRs in the central region of intrafusal fibers.

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 by in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 by insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but, unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.