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All-women cyclathon to promote healthy lifestyle and breast cancer awareness – The Statesman

The Bhubaneswar Smart City Limited (BSCL) on Saturday organized an all-women cyclathon to promote a healthy lifestyle and creating awareness on breast cancer, with partnering civil society organizations, KRIAA Foundation and Commissionerate of Police.

The rally formed part of the observance birth anniversary of the father of the nation-Mahatma Gandhi and former Prime Minister Lal Bahadur Shastri.

The cyclathon started at 7.05 am from Sishu Bhawan Square and culminated there at 7.50 am by passing through Janpath, Exhibition Ground Road, Sachivalaya Marg and Udhayn Marg. The participants are from Rama Devi Womens University and Kamala Nehru Womens College, respectively.

The event was presided by Director Acharya Harihar Post-Graduate Institute of Cancer, Cuttack Professor Dr Lalatendu Sadangi. Leading surgeon and Professor and HOD of Surgical Oncology in AIIMS Bhubaneswar Dr Madhabananda Kar joined as the guest of honour. General Manager Bhubaneswar Smart City Limited Kamaljit Das and founder of KRIAA Foundation Lili Jenamani also spoke on the occasion.I am very happy to participate in the event in which we all participants used the Mo Cycle and enjoyed the ride. With the city providing a beautiful atmosphere to the cyclists, we all enjoyed the event, said

The event was well-organised to provide a unique platform and the awareness of a prominent health issue of women. We also got an opportunity to check the cycling infrastructure of the city and found that it is very much citizen-friendly, observed another woman participant Muzda Taliha.

Bhubaneswar has developed a good city-wide facility for cycles and pedestrian ways so that the emissions are less and citizens enjoy a healthy environment to breathe in the fresh air with fewer pollutants and particulate matter in the neighbourhood air. The city has relied on its non-motorized transport (NMT) planning to make it more livable, the officials claimed.

We are committed to providing better and healthy living conditions and facilities for citizens so that the clean and green tag of Bhubaneswar would be seen translated to reality in every zone and subzones to make it a wonderful place to live in, Managing Director and Chief Executive Officer, Bhubaneswar Smart City Limited Sri Sanjay Kumar Singh said while speaking on the occasion.

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Is Male Menopause a Real Thing That Guys Deal With? – AskMen

Ah, middle-age the self-doubt, the existential angst, the sudden awareness of just how short life is. Work, marriage, kids. Its not easy. And then comes the lack of enthusiasm, the tiredness, the brain fog and the irritability. But these are not necessarily de facto products of having made it to a certain age.

Rather they aresymptoms and you can throw in hot flushes, insomnia, mood swings and a general disinterest in sex of a still controversial idea: that, like women, men experience menopause.

This is not a definitive biological, hormonal event as it is for women the sometimes decade-long spell of similar symptoms, leading to a lack of menstruation for a year, followed by officially entering ones post-child-birthing phase of life. It likely doesnt have quite the same negative impact on well-being, on relationships and work.

Certainly not everyone in medical circles thinks its even a real thing, and scientific study of the manopause more officially, the andropause is lacking. And yet many men, and increasingly, clinicians too, concede that something sometimes happens when you hit your mid-40s or early 50s. You go off the boil.

RELATED:Turning 30? Here's How Your Bod's Going to Change

The male menopause just isnt as well recognized as the female menopause, explains Dr. Clive Morrison of Londons Centre for Mens Health. This is in part because the latter is a natural, well-documented event for every woman, but its a less distinct, drawn-out process for most men, and it doesnt affect all men either. In part its because were not really sure why it happens to men. And in part also because of culture, which still broadly has a temperance attitude to sex. The male menopause is confused with the stigma around loss of libido, even though thats only one possible symptom. But this is more about your mojo. Its about your quality of life.

It doesnt help, of course, that a lot of the many possible symptoms of andropause are products of aging too: like it or not you can start to lose muscle typically about 3-5 percent of your muscle mass every 10 years after the age of 30. You might put on weight too, or rather your body fat is redistributed, so you get a bit of a belly. This all makes correct diagnosis of the andropause tricky a situation only made worse because, as Morrison stresses, each of us has our own threshold levels for each symptom, such that a man may display all of the psychological signs and some but by no means all of the physical or bio-chemical ones.

And psychological factors may be indicative of something else depression, maybe, not the andropause. Inevitably this all makes deciding on the right course of action tricky too.

There are simple things that we know do help that, in fact, help towards better health generally. Move more at a minimum just taking good, regular, long walks. Train gently but regularly with weights. Eat a low-carb, high protein diet. Get a good eight hours sleep every night. Address the stress in your life rather than normalizing it.

But others those who may be tempted to supply a quick fix, much as HRT or Hormone Replacement Therapy has (not uncontroversially) become for menopausal women also suggest that the andropause is likewise a hormonal issue in men. Its all, they say, about a loss of testosterone, as for womenabout estrogen.

Indeed, far from the misconception of testosterone as being all about sex, its decline is also linked to muscle atrophy, reduced bone density and a susceptibility to depression; and theres an inverse correlation with obesity and mortality from heart failure the less of it you have, the more prone you are to both issues. This is why some experts dub the male menopause Testosterone Deficiency Syndrome. And why often testosterone replacement therapy is pushed.

Anecdotally, that certainly works for some men. But dont rush into this. For one, while the rapid shutdown in estrogen production has been attributed to symptoms of the female menopause, men typically see only a 2% testosterone decline per year over their thirties. Its argued that this is unlikely to have noticeable consequences.

Then there are the practical issues: regular testosterone injections this is how its typically delivered into the bloodstream dont come cheap. Prices vary but were often talking at least a few hundreds dollars per month and, obviously, those who recommend this treatment also profit from it too. Nor can you just have these as boosters to get back on track: since injecting testosterone also typically causes your natural testosterone production to gradually close down, once you start, youre committed for life. Its a crutch, not a cure.

RELATED:Heres the Healthy Heart Advice You Aorta Follow

This doesnt help when a drop in testosterone can be hard to measure conclusively healthy testosterone levels may disguise a decline from even higher ones. Current understanding also has it that subtle shifts in testosterone levels are more part of a broader package of metabolic changes in various key hormone levels prolactin, gonadotropin, DHEA and others youve likely never heard of with consequences similar to those experienced by menopausal women.

Its all very complex, sighs Morrison. Its often partners who first notice the problems the moodiness, the low libido. But then that gets misinterpreted and the man is sent off for marriage guidance when that kind of intervention isnt whats needed at all. Its the male menopause.

But understanding that, understanding the part played by testosterone which a lot of general practitioners still think of as some kind of snake oil is only recent. Thats just over the last 10 to 20 years, which unfortunately is not very long in medicine.

In other words, were still some way from grasping quite what the andropause is, what to do about it and, in some quarters, whether theres anything to do anything about. All of which is little consolation for those men who are suffering from a nasty potpourri of physical and psychological ailments that just when careers are peaking, just when theyre maybe wrangling both children and elderly parents, when retirement seems a lifetime away and time to look after themselves is almost zero comes along and really screws with their day.

But dont let that put you off. If youre not feeling right, and something inside leaves you unconvinced if its all just the normal pressures of a certain time of life, see a doctor. The manopause may sound like a joke, but it doesnt feel like it.

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10 Warning Signs That You May Have Low Testosterone – News18

Sex may permeate our popular culture, but conversations about it are still associated with stigma and shame in Indian households. As a result, most individuals dealing with sexual health issues or trying to find information about sex often resort to unverified online sources or follow the unscientific advice of their friends.

To address the widespread misinformation about sex, is running this weekly sex column, titled Lets Talk Sex, every Friday. We hope to initiate conversations about sex through this column and address sexual health issues with scientific insight and nuance.

The column is being written by Sexologist Prof (Dr) Saransh Jain. In todays column, Dr Jain explains low testosterone or (low T), its signs, symptoms and treatment options.

Testosterone is a hormone produced in both male and female bodies. In men, its produced in the testicles and affects several different physical features and functions. Testosterone in men stimulates sex drive, sperm production, and development of muscle and bone mass. It is also responsible for hair growth, causes the voice to deepen, among other male features.

After the age of 30, testosterone levels in men can decline somewhat, but this is not a problem. But if your testosterone levels get too low, it can cause a range of symptoms and interfere with your daily life, overall wellness, and relationships.

The signs and symptoms of low testosterone are sometimes overlooked, attributed to other causes or ignored due to the condition itself.

Extra Body Weight

Its not clear if low testosterone (low T) contributes to weight gain, or if weight gain contributes to low testosterone. However, fat cells play a role in converting testosterone to estrogen, the dominant female sex hormone. Healthy eating and exercise, therefore, are must.

Loss of Muscle

While testosterone doesnt affect the function or strength of your muscles, its necessary for building muscle mass. If you find youre losing muscle volume, it may be due to lower levels of testosterone affecting new tissue growth as well as existing muscle maintenance.


Are you tired all the time? It might not just be about getting older or mounting stress at work. Perhaps you find it hard to get yourself motivated to exercise, or you dont feel rested after a full nights sleep. While there are many potential causes of fatigue, add low T to the list, particularly if you have other symptoms.


Although commonly associated with older women as a side effect of estrogen loss, loss of bone mass in men results from low testosterone levels. The results are the same. Bones may fracture or compress more easily because of an increasingly porous structure.

Low Sex Drive

This is a condition that many ignore, since in some cases you wont feel the absence of your libido, even though its gone. Testosterone is a key factor in triggering sex drive in men and women, when its level drops, so may your desire to have sex.

Erectile Dysfunction

Testosterone stimulates the brain to produce nitric oxidea molecule that triggers the chemical reactions necessary for an erection. Low testosterone can make it difficult for men to maintain or achieve an erection. There are other common factors that can lead to erectile dysfunction. Some include thyroid issues, smoking, stress, alcohol and tobacco consumption, high blood pressure and diabetes. However, low testosterone is another common cause for erectile dysfunction.

Low Volume of Ejaculation

Low testosterone causes a reduction in the amount of semen your body manufactures. When this occurs, the amount of semen released during orgasm may be less than the amount youre accustomed to. Again, in combination with other signs, low semen volume may indicate low testosterone production.

Hair loss

While there are hereditary factors that influence hair loss, low testosterone also contributes to it, and its the likely culprit if youre losing body and facial hair as well.

Changes in Sleep Patterns

Decreased energy, trouble sleeping, and even sleep apnea have been associated with low testosterone.

Depression and Mood Disorders

Over 50 per cent men in a study on the effects of low testosterone also showed signs of depression, and depressed men often find that testosterone replacement therapy can sometimes be more effective than antidepressants.

If you have any or all of these symptoms of low testosterone, a simple blood test can establish your T levels. Combined with your medical history, your blood test can suggest why your testosterone levels are low. The underlying cause of low testosterone can determine your treatment options.

What you eat has a major impact on testosterone as well as other hormone levels. Eating enough protein can help maintain healthy levels and aid fat loss, which is also associated with your testosterone. A diet based mainly on whole foods is best, with a healthy balance of fat, protein and carbs, which can help boost testosterone levels.

If you do experience low testosterone symptoms, your healthcare provider may order a blood test to measure your total testosterone level, luteinizing hormone (LH), blood prolactin level, and blood hemoglobin or HgB.

Testosterone Therapy (or TT) is sometimes prescribed if an individual has a testosterone deficiency. Typically, there are five different ways in which this can be done:

Skin patch: A patch is applied once every 24 hours, in the evening, which releases small amounts of the hormone into the skin.

Gels: Topical gels are spread daily onto the skin over both upper arms, shoulders, or thighs. It is important to wash your hands after applying and to cover the treated area with clothing to prevent exposing others to testosterone.

Oral therapy: Capsules are swallowed or tablets are attached to your gum or inner cheek twice a day. Testosterone is then absorbed into the bloodstream.

Pellets: These are implanted under the skin, usually around the hips or buttocks, and slowly release testosterone. They are replaced every three to six months.

Injections: Various formulations are injected every seven to 14 days. Testosterone levels can rise to high levels for a few days after the injection and then slowly come down, which can cause a roller-coaster effect, with mood and energy levels spiking before trailing off.

If you are concerned about low-T symptoms, or if youve tried therapies that didnt work, talk to your primary doctor or sexologist. Personalized care from a board-certified sexologist or male fertility expert is the healthiest way to get there.

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Global Osteoporosis Drug Market Size and Analysis, Services With Its Application and Access Type by Future Forecast 2021-2028 EcoChunk – EcoChunk

DBMR has added another report named Global Osteoporosis Drug Market with information Tables for recorded and figure years addressed with Chats and Graphs spread through Pages with straightforward definite examination. The a-list report concentrates on broad assessment of the market development expectations and limitations. The systems range from new item dispatches, extensions, arrangements, joint endeavors, organizations, to acquisitions. This report includes profound information and data on what the markets definition, characterizations, applications, and commitment and furthermore clarifies the drivers and restrictions of the market which is gotten from SWOT investigation. Worldwide market examination report serves a great deal for the business and presents with answer for the hardest business questions. While making Global Osteoporosis Drug Market report, examination and investigation has been completed with one stage or the mix of a few stages relying on the business and customer necessities.

Market definition canvassed in the predominant Global Osteoporosis Drug Market advertising report investigates the market drivers that show factors causing ascend in the market development and market limitations which demonstrate the components causing fall in the market development. It helps clients or other market members to know about the issues they might confront while working in this market throughout a more extended timeframe. This statistical surveying report additionally concentrates on utilization of market, central participants included, deals, value, income and portion of the overall industry with volume and an incentive for every area. The greatness and straightforwardness proceeded in business research report makes acquire the trust and dependence of part organizations and clients.

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Global Osteoporosis Drug Market By Type (Primary Osteoporosis and Secondary Osteoporosis), Therapy Type (Hormone replacement therapy and Bisphosphonate therapy), Treatment Type (Medication and Surgery), Mechanism of Action Type (Bisphosphonates, Selective Estrogen Receptor Modulators and Bone Resorption Inhibitors), Route of Administration Type (Oral, Intravenous, Subcutaneous), End- Users (Hospitals, Homecare, Specialty Clinics, Others), Geography (North America, South America, Europe, Asia-Pacific, Middle East and Africa) Industry Trends & Forecast to 2026

Global Osteoporosis Drug Research Methodology

Data Bridge Market Research presents a detailed picture of the market by way of study, synthesis, and summation of data from multiple sources. The data thus presented is comprehensive, reliable, and the result of extensive research, both primary and secondary. The analysts have presented the various facets of the market with a particular focus on identifying the key industry influencers.

Major Drivers and Restraints of the Osteoporosis Drug Industry

Increases prevalence of osteoporosis worldwideVulnerable aging population of menopause womenEmergence of drugs used in the treatment of celiac disease, inflammatory bowel disease, hyperthyroidism, chronic obstructive pulmonary disease (COPD) and bone marrow cancerAccelerates demand of novel therapies and newer development

Strategic collaboration and licensing deal between the companiesRising awareness about treatment and technological advancement is driving the growth of marketEffective treatment is either unavailable or unaffordable.Patent expiry from many companies and introduction of generic drugs of branded version is expected to restrain the growth if the marketInadequate knowledge about osteoporosis in some developing countries

Complete report is available (TOC) @

The titled segments and sub-section of the market are illuminated below:

By Type

Primary OsteoporosisPostmenopausal OsteoporosisSenile OsteoporosisIdiopathic OsteoporosisSecondary Osteoporosis

By Therapy Type

Hormone replacement therapy (HRT)Testosterone replacement therapyEstrogen therapyBisphosphonate therapy

By Treatment Type

MedicationCalcium and Vitamin D supplementsAntacidsSurgeryVertebroplastyKyphoplastyOthers

By Mechanism of Action Type

BisphosphonatesAlendronateIbandronateRisedronateZoledronicSelective Estrogen Receptor Modulators (SERMs)RaloxifeneBone Resorption InhibitorsDenosumab

By Route of Administration


By End Users

HospitalsHomecareSpecialty ClinicsOthers

Top Players in the Market are:

Few of the major competitors currently working in the global osteoporosis drug market are Allergan, Amgen Inc, Astellas Pharma Inc, AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company, Teijin Pharma Limited, Stelis Biopharma, Radius Health, Inc, Pfizer Inc, Ono Pharmaceutical Co., Ltd, Novartis AG, Mochida Pharmaceutical Co., Ltd, Mereo BioPharma Group plc, Merck & Co., Inc, Gedeon Richter (UK) Ltd, F. Hoffmann-La Roche Ltd, Novo Nordisk A/S, Cipla Inc, UCB SA, Teva Pharmaceutical Industries Ltd and among others.

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Some of the Major Highlights of TOC covers:

Chapter 1: Methodology & Scope

Definition and forecast parameters

Methodology and forecast parameters

Data Sources

Chapter 2: Executive Summary

Business trends

Regional trends

Product trends

End-use trends

Chapter 3: Osteoporosis Drug Industry Insights

Industry segmentation

Industry landscape

Vendor matrix

Technological and innovation landscape

Chapter 4: Osteoporosis Drug Market, By Region

Chapter 5: Company Profile

Business Overview

Financial Data

Product Landscape

Strategic Outlook

SWOT Analysis

Thanks for reading this article, you can also get individual chapter wise section or region wise report version like North America, Europe or Asia.


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An absolute way to forecast what future holds is to comprehend the trend today!Data Bridge set forth itself as an unconventional and neoteric Market research and consulting firm with unparalleled level of resilience and integrated approaches. We are determined to unearth the best market opportunities and foster efficient information for your business to thrive in the market. Data Bridge endeavors to provide appropriate solutions to the complex business challenges and initiates an effortless decision-making process.

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Eddie Hall Says Going Vegan Made His Body Wither Away – Men’s health UK

It's fair to say that Eddie Hall is a man who likes his food. During his strongman career, he would consume over 12,000 calories a day. Even now, being leaner and smaller, Hall still puts away between 5000 and 7000 calories every day.

But speaking exclusively to Men's Health UK, Hall revealed that there's no chance he would ever consider getting all of those calories from a vegan diet and revealed how he felt like his body was "withering away" during a short experiment with veganism.

"I've tried it in the past and you just see your body withering away, you just can't sustain it," says Hall. "At the end of the day I'm a 156 kilo man, I'm one of the biggest men in the country, and you're not going to maintain that being vegan. That would put a massive hindrance on my performance."

Hall says that when he tried the diet "for like three or four days" he felt his strength and size was falling away. Admittedly, it's not a huge amount of adaptation time, but "it just didn't suit me," he says.

Still, Hall is keen to point out that he's not against veganism altogether. His wife eats a lot of vegan meals, and is probably best described as a flexitarian. Hall just thinks that while it's good to reduce the amount of meat you eat, it's hard to maintain strength and muscle on a diet that includes no meat at all.

"Although there's a lot of good things about being vegan, for me to maintain the strength and power that I need, I just think it's impossible to keep that size and strength and be a vegan, so I have to get my meat in me for the protein side of things, the amino acids and everything," says Hall.

"I'm a big believer in less meat is good for you, but cutting meat out completely is a big no no."

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How Veganism Is Rooted in Black Activism, and Why It Isnt Just For White People – POPSUGAR

Contrary to who gets visibility within the vegan community, Black people make up the fastest growing vegan demographic. With social media influencers like Tabitha Brown reshaping the narrative on the importance of representation and racial diversity within this community, veganism is well on its way to becoming more inclusive. However, as veganism gains more popularity in the mainstream media, many often forget to acknowledge the long history of Black veganism which is centrally tied to Black activism of the 1960s as well as the African roots of plant-based diets.

The racial reckoning of 2020 has unearthed a necessary conversation on the need for more inclusivity within the vegan community, one that has and continues to be dominated by white women. Even Brown has said that she thought vegans were "white women who did yoga." However, the uplifting of Black vegan creators who were previously sidelined in the community has brought more awareness to the activist roots of veganism.

The late comedian Dick Gregory was an influential activist during the Civil Rights Movement. Not only did he advocate for the Black community, but he also protested the Vietnam War and was very outspoken about his choice to not eat meat instead choosing a plant-based diet as a form of activism. Gregory denounced the killing of humans and animals in his 1971 food manifesto titled, Dick Gregory's Natural Diet For Folks Who Eat: Cookin' With Mother Nature.

During an interview on Studs Terkel's radio show, Gregory credited Dr. Martin Luther King Jr. for inspiring him to change his diet, and brought attention to how veganism and nonviolent protests are intrinsically linked. Gregory's famous quote, "Don't be wearing no leather shoes," became a rallying cry for social issues, specifically about not consuming animal products. Many people, inspired by Gregory's actions, have come to view veganism as a way to fight oppression from harmful foods that are marketed to and oversaturated in Black neighborhoods across the country.

The fight for racial justice continues, and plant-based diets are now being seen as essential tools to combat the systemic inequities that have persisted over generations. At the heart of veganism are African plant-based diets. Prior to colonization, the diet of our ancestors consisted of yams, greens, vegetables, and beans meals containing no dairy, eggs, or meat. Veganism is a return to the traditions of an African plant-based diet, which will positively impact our health and longevity.

White people have largely been the ones to profit off veganism. They can now help uplift Black vegans by learning more about veganism's roots in the Black community and finding meaningful ways to give back to these under-resourced communities. I hope that others who see veganism as a trend, fad, or something to appropriate understand that for our community, food has and always will be interconnected to Black identity, culture, struggle, resistance, and triumph.

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Slutty Vegan Kicks off PETA’s Food Justice Campaign with Free Meals – One Green Planet

PETA teamed with Atlanta restaurant, Slutty Vegan, to kick off their food justice campaign that brings awareness to the issue of food deserts.

The campaign calls on government officials to stop focusing so much on meat, eggs, and dairy in food deserts, and to instead provide fresher and more humane options, like fresh produce.

Together PETA and Pinky Cole from Slutty Vegan kicked off their campaign in Atlanta by handing out free entrees and vegan meal starter kits from PETA.

Cole said, Weve made veganism fun and accessible at Slutty Vegan and are all about indulging in the little pleasures life brings. We want to show that eating plant-based doesnt have to be boring. With lines down the block at each of our locations, were honored to bring good, kinder food to Atlanta and are grateful for PETA taking up this issue on a national level.

Atlanta is just the first stop for this campaign. The powerful vegan team will also be visiting Baltimore, Indianapolis, Los Angeles, and other cities.

Every year the US government spends $38 billion in tax money on the meat, egg, and dairy industry. In comparison, it only spends $17 million on the produce industry, despite scientific evidence that shows the importance of eating plant-based foods. Those in food deserts suffer all the more for this as they have very few accessible and healthy options.

For more Animal, Earth, Life, Vegan Food, Health, and Recipe content published daily. Subscribe to the One Green Planet Newsletter! Lastly, being publicly funded gives us a greater chance to continue providing you with high-quality content. Please consider supporting us by donating!

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Groping toward appropriate regulation of AI. Data regulation and its offensive potential. – The CyberWire

At a glance.

Wired traces the influences shaping the EUs proposed Artificial Intelligence Act, which is expected to impact policy internationally, as has the GDPR. The legislation would categorize AI applications by risk level and more closely control high risk systems.

Critiques of the bill fall along predictable lines. Some human rights groups want stricter controls and worry about law enforcement, education, health care, public surveillance, border security, social scoring, insurance, transhumanism, and subliminal manipulation applications. They point to the power disparity between those wielding the tools and those on the receiving end, and highlight existing abuses.

Some industry groups describe the law as overbroad, fearing it will impose unmanageable costs, interfere with basic business functions, squash innovation, and drive away talent. Competing studies put total compliance costs between 1.6 and 10 billion yearly.

The EU hopes the bill will level the playing field and spur growth while promoting principled business decisions. Meanwhile, the US is developing its own guidelines and regulations, including a National Institute of Standards and Technology tool and an Algorithmic Accountability Act. In the background, as always, looms Chinese innovation, and what strategic advantages authoritarian rivals will achieve while the West puzzles out competing interests and ethical dilemmasexercising a soft advantage of its own.

Breaking Defense reiterates concerns that the vulnerability disclosure component of Beijings Data Security Law (DSL) will help the CCP stockpile zero days for use against state and private sector targets. As weve seen, the legislation directs researchers, companies, and foreign firms with local offices to disclose to the Ministry of Industry and Information Technology within forty-eight hours uncovered zero days, and restricts their further distribution. Microsoft and Amazon Web Services, both of which contract with the US Defense Department, are covered by the regulation.

Heritage Foundation China scholar Dean Cheng sees the move as an instance of lawfare, or legal warfare, and says Beijing is 100 percent likely to weaponize the disclosed vulnerabilities. Georgetown University security researcher Dakota Cary observed that theyve effectively co-opted a pipeline of research, which costs a great deal of money to do, in order to increase their own offensive and defensive hacking capabilities.

The DSL, Fortune notes, also prohibits unapproved cross-border data transfers. The law took effect yesterday.

SWI reports that Switzerland is working to establish a rapid reaction cyber defense command center staffed by roughly six-hundred military personnel with new capacities to protect private sector and critical infrastructure assets. The center will deliver informational, logistical, and technical capabilities.

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Groping toward appropriate regulation of AI. Data regulation and its offensive potential. - The CyberWire

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From the journals: MCP – American Society for Biochemistry and Molecular Biology

Discovering drugs in deadly venom. Improving transplant outcomes with better storage. Linking mannose glycosylation to eye development. Read about papers on these topics recently published in the journal Molecular & Cellular Proteomics.

The purple cone snail, Conus purpurascens, hunts fish and uses venom to immobilize its prey. Cone snail venom contains diverse toxic peptides, or conopeptides, that are biologically active, target-specific and valuable for drug discovery. One of the most powerful known painkillers, ziconotide, marketed as Prialt, was derived from a conopeptide.

Alex Holt/NIST

A purple cone snail uses its harpoon to pierce through a latex-covered tube,allowing a researcher to collect its venom.

Conopeptides vary among the more than 800 cone snail species and among members of the same species. Even a single cone snail specimen can produce unique conopeptide cocktails, called cabals, specialized for predation or defense. Cone snails also can hypermodify conopeptides at the post-translational step, increasing the diversity of the toxins and extending their range of biological targets. The extreme diversity of conopeptides provides a rich source of biologically active molecules for drug discovery.

In a recent study in the journal Molecular & Cellular Proteomics, Meghan Grandal and colleagues at the National Institute of Standards and Technology collected and analyzed the injected venom from 27 specimens of the purple cone snail. Using high-resolution mass spectrometry techniques, they discovered 543 unique conopeptides derived from 33 base peptide sequences 21 of which were newly identified base peptides. An abundant and newly discovered conopeptide named PVIIIA illustrates the complexity and diversity of modifications to the base conopeptide that occur among various purple cone snail specimens. Building on previous studies, the researchers showed that the different snail specimens produce one of two unique venom cocktails. These two cocktails correspond to what are known as the lightning strike cabal that rapidly induces paralysis of the snails prey and the motor cabal that acts more slowly to induce irreversible paralysis.

Knowing which conopeptides are co-expressed within a specific cocktail will give the researchers important clues as to the possible neural targets of newly identified conopeptides. This will be a critical step in developing new conopeptides into neural probes or therapeutics.

The demand for kidney transplants exceeds the supply of available kidneys. Some donated kidneys go unused, however, due to the prolonged time between circulatory arrest and the start of cold storage. These kidney graftsoften fail or are slow to function. Repairing such kidneys before transplant could greatly increase the available supply.

An unusual type of protein glycosylation, C-mannosylation, involves attaching a single mannose sugar to the amino acid tryptophan by a carboncarbon bond. C-mannosylation, which regulates protein secretion, folding and function, occurs at a specific sequence of four amino acids that begins with the modified tryptophan. Even though about 18% of secreted or transmembrane proteins have this sequence, few studies have looked for the modification. Consequently, researchers know of few proteins that are C-mannosylated.

In a new study in the journal Molecular & Cellular Proteomics, Karsten Cirksena of the Institute of Clinical Biochemistry and a team of researchers in Germany found numerous proteins with altered secretion levels in cells lacking the C-mannosylation machinery. One of these potentially C-mannosylated proteins, a disintegrin and metalloprotease with thrombospondin motifs, or ADAMTS16, is essential during eye development and optic fissure closure. In Chinese hamster ovary cells and Japanese rice fish, the researchers demonstrated that ADAMTS16 can be C-mannosylated, that its secretion depends on C-mannosylation and that loss of a C-mannosylation enzyme causes a developmental eye defect known as a partial coloboma a gap in the eye tissue. Their findings suggest that C-mannosylation, an understudied protein modification, plays a critical role in eye development by regulating secretion of ADAMTS16.

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Stress increases in exopher-mediated neuronal extrusion require lipid biosynthesis, FGF, and EGF RAS/MAPK signaling –

Proteostasis is critical for the function, maintenance, and long-term survival of all cells (1). Successful maintenance of the proteome entails efficacious balancing of protein synthesis and degradation, with coordinated actions of regulated gene expression and translation, the chaperone-folding network, the ubiquitin proteasome system, and autophagy all serving critical functions in overall protein quality control. Neurons are particularly susceptible to proteostasis disruption, and aggregates are a striking and common feature of neuropathology in most human neurodegenerative diseases (2). Recent studies have shown that in diseases characterized by distinctive aggregates, including Alzheimers disease, Parkinsons disease, and Huntingtons disease, aggregates can spread from neurons to neighboring cells, inducing deleterious consequences (3). The mechanisms operative for aggregate spread are unclear and remain a matter of considerable research attention.

We discovered that adult Caenorhabditis elegans neurons can select and then extrude aggregated proteins, such as expanded polyglutamine HTTQ128-CFP (related to neurotoxic Huntingtons disease protein) and an aggregation-prone, high-expression mCherry reporter (4) (see Fig. 1A). Aggregates are sent out of the neuron in strikingly large, membrane-surrounded vesicles [4-m average diameter; for comparison, the neuron soma is 6 m; this vesicle is two orders of magnitude larger than exosomes that form by a distinct mechanism (5)]. We refer to these massive cell extrusions as exophers (exo = outside and pher = carry away). In the case of the C. elegans touch receptor neurons that mediate sensitivity to gentle touch stimuli (6), exophers are passed into the surrounding hypodermis, which can partially degrade their contents. Baseline production of exophers under nonstress growth conditions is low, detected in 5 to 20% of ALMR touch neurons, and occurs at lower rates for other touch neurons. However, we found that exacerbated proteostress (for example, expressing the human A142 fragment implicated in Alzheimers disease pathology, disrupting specific autophagy genes via RNA interference [RNAi] or introducing the MG132 proteasome inhibitor) can significantly increase exopher production (4). Animals expressing toxic HTTQ128-CFP in touch neurons that extruded aggregates in exophers retained better touch sensitivity than transgenic HTTQ128-CFP animals in which the neurons did not produce exophers (4), suggesting that exopher production that helps clear the neuron of toxic aggregates is neuroprotective, at least in the short term.

A key mystery in the biology of exophers is the precise nature of the cellular conditions and stresses that induce or elevate exophergenesis. To address this question, we systematically tested external, physiological stresses for the capacity to influence the expulsion of cellular trash. Our data 1) show a clear link between specific, environmental stresses and neuronal exopher production (namely food withdrawal, osmotic stress, and oxidative stress); 2) emphasize that there exists a permissive window for exopher production in early adult life during which external stresses can elevate exopher levels; and 3) identify a previously unrecognized limit to the level of stress that can induce exophers, such that, beyond this upper stress limit, exophergenesis is not observed. We linked the fasting-induced increase in exophers (as much as 10-fold elevation in exophers) to activities of the intestinal peptide transporter PEPT-1, the transcription factors MDT-15 and SBP-1/SREBF2, and their target FASN-1/fatty acid synthase, as well as to epidermal growth factor (EGF) and fibroblast growth factor (FGF)/RAS/MAPK signaling pathways. Our data reveal how stress conditions might promote the spread of aggregates from neurons to their neighbors and suggest pathways that might be targeted to regulate analogous processes, with implications for addressing human neurodegenerative disease.

A common stress that C. elegans encounter in their natural environment is variable food type and/or food abundance, which can have a significant impact on C. elegans gene expression, development, metabolism, and longevity (79). In the laboratory, C. elegans eat a diet of Escherichia coli spread on agar plates, and food sources can be easily manipulated. We first asked whether neuronal exophergenesis levels are sensitive to food source, quantitating ALMR exophers produced by neurons expressing an mCherry protein (strain ZB4065 bzIs166[Pmec-4mCherry1]), which is avidly expelled as exopher cargo (10) (see Fig. 1 for an example). For claritys sake we refer to assay strain ZB4065 as mCherryAg2 in the text hereafter. Exophers are typically produced by young adult animals, peaking at days 2 to 3 of adulthood and returning close to baseline by adult day 4 (Ad4) (4, 11). We therefore measured exophers produced each day, Ad1 to Ad4, to compare both daily relative levels and temporal profiles.

We quantitated exopher production when mCherryAg2 animals were fed four different E. coli strains: E. coli OP50, which is the standard C. elegans food source (12); HT115, a strain that lacks RNAaseIII and is used in RNAi studies (13); HB101, a food source that promotes larger body size and faster development (14); and NA22, a strain that fosters enhanced growth in liquid culture (15). We noted no major differences among the different E. coli strains in common laboratory use, all of which support the basic pattern of peak exopher production around Ad2 that returns to the baseline around Ad4 (SI Appendix, Fig. S1A).

Complete food withdrawal markedly induces exophers. We reared mCherryAg2 animals at 20 C and at Ad1 (onset of egg laying) moved animals to unseeded plates at 20 C for either 3 or 6 h, counting ALMR exophers shortly thereafter. Continuously fed control mCherryAg2 animals generate exophers at levels that range from 5 to 20% of animals, and in this study, baseline was close to 5% on Ad1. Subsequent to a 3-h fast, we found exophers in more than 20% ALMRs; a 6-h fast increased average exopher numbers 5- to 10-fold to nearly 50% of ALMRs examined (Fig. 1B). Fasting also causes an increase in multiple-exopher events, in which more than one exopher is generated (SI Appendix, Fig. S1B). We conclude that food withdrawal can have a rapid and dramatic impact on the extrusion of exophers in animals expressing a noxious mCherry reporter.

At 20 C, in the presence of food, the young adult wave of exopher production falls narrowly within the first 4 d of adulthood (SI Appendix, Fig. S1A). We were therefore curious as to whether the fasting-induced elevation of exophers was restricted to the Ad1 to Ad4 timeframe or, alternatively, whether food withdrawal experienced at any time point could be effective for inducing exopher production. We subjected animals from a single, synchronized population to 6-h fasting regimens as L4 larvae as well as on Ad1 to Ad10; we measured exopher levels immediately after the 6-h fast on the experimental fasting day (Fig. 1C). Our analysis of mCherryAg2 ALMR exophers in this study revealed the following: 1) a 6-h fasting protocol does not induce exopher production during the L4 stage; 2) the 6-h fasting protocol elevates exopher production to peak levels at Ad1 when administered on that day; 3) fasting efficacy is slightly lower when delivered at Ad2 and lower still when delivered at Ad3; and 4) by Ad4, a fasting-induced exopher increase is no longer observed. We conclude that the impact of food withdrawal on enhancing exopher production is limited to a window of time that covers Ad1 to Ad3. The flanking of the permissive phase by exopher-recalcitrant periods suggests the existence of a licensed period, in which the expulsion of significant amounts of neuronal material is biologically feasible and during which exophergenesis can be modulated by environmental stress signals. We observed the 6-h fastinginduced elevation of exophers in strains that expressed GFP (Fig. 1D) or expanded polyglutamine (Fig. 1F) mNG:HttQ74 in touch neurons or expressed GFP in dopaminergic neurons (Fig. 1E), indicating that the fasting induction of exophers was not reporter- or cell typespecific.

Temperature is an environmental factor that dramatically influences the C. elegans reproductive life cycle time, lifespan (16), and proteostress (17). We therefore asked whether culture temperature might influence the timing or levels of adult exopher production. We reared animals continuously at 15, 20, and 25 C from the egg stage and measured exopher levels over the first 4 d of adult life (SI Appendix, Fig. S2A). Somewhat unexpectedly, exopher levels did not vary substantially with culture temperature. In an alternative experimental design, we reared synchronized populations of animals at 20 C and then split cultures at the L4 adolescent stage into three parallel cultures that were thereafter maintained at 15, 20, and 25 C (SI Appendix, Fig. S2B). We found that, in animals shifted to 25 C, the peak of exopher production occurred earlier (Ad1) than 20 C animals, but exopher levels were not significantly elevated, relative to 20 C. Our data suggest that the experience of a temperature shift, rather than continuous noxious temperature exposure, might exert the strongest impact on levels of exopher production (SI Appendix, Fig. S2C). That said, the approximate doubling of exopher scores after an L4 shift from 20 to 25 C is modest in comparison to the 5 to 10 exopher elevation measured for food withdrawal. We conclude that, within the normal confines of C. elegans laboratory culture, the temperature at which animals are grown exerts a relatively minor influence on levels of exopher production.

Hypoxia can induce protein aggregation in C. elegans models (18), raising the possibility that hypoxia might enhance exopher production. We tested the exposure to 0.1% oxygen using an adjustable hypoxia C-174 chamber (Biospherix) to generate exposures to a controlled, hypoxic environment. We subjected Ad1 to Ad5 mCherryAg2 animals to 0.1% oxygen for 6 h and monitored ALMR exophers after the removal of animals from the chamber.

We failed to find differences in exophers produced under these hypoxic conditions on any day (SI Appendix, Fig. S2D). We also tested anoxic conditions induced by replacing oxygen with nitrogen in a sealed anoxia chamber (19), measuring ALMR exophers after 6-h exposure to anoxia (SI Appendix, Fig. S2D, dark purple line). Exposure to anoxia failed to markedly increase exopher production on any exposure day. While it is possible that longer exposures or different recovery times might alter outcomes, we conclude that hypoxia/anoxia treatments, as delivered, do not elevate exopher levels.

Osmotic stress introduces proteostasis challenges (20), and thus, we were curious as to how exopher production might respond to osmotic stress conditions. We first made agar plates designed to introduce osmotic stresses based on standard C. elegans conditions, testing 250 mM concentrations of sucrose, glucose, sorbitol, and NaCl. At Ad1, we introduced mCherryAg2 animals to these osmotic stresses for 6 h and scored ALMR exophers thereafter (Fig. 2A). We found that a 6-h exposure to osmotic stress increased exopher production 4 above baseline. Since each solute-induced stress resulted in a similar elevation of exophers, the exopher response is likely grounded in osmotic stress itself, rather than the chemical nature of the specific osmolytes.

Osmotic stress and oxidative stress can increase exopher production, but extreme stress levels can decrease exophergenesis. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) Transient osmotic stress significantly elevates ALMR exophers on Ad1. We exposed mCherryAg2 animals to 250 mM sucrose, glucose, sorbitol, or NaCl on Ad1 and scored for exophers 6 h later: 5 trials and 50 animals per trial. (B) Animals chronically exposed to osmotic stress from Ad1 into adulthood produce elevated exophers with early onset peak production. We grew mCherryAg2 animals to the Ad1 stage under standard growth conditions and shifted to 250 mM sucrose, glucose, sorbitol, or NaCl, measuring ALMR exophers in these populations on Ad1 to Ad4: 4 trials and 50 animals per time point. CMH compares osmotic stress media to normal growth media. (C) Osmotic stress at 250 mM enhances exopher production more than at 500 mM. We exposed strain mCherryAg2 to 250 and 500 mM sucrose, glucose, sorbitol, and NaCl for 6 h on Ad1 and measured exophers shortly thereafter: 5 trials and 50 animals per trial. (DF) Increasing oxidative stress enhances exopher production to a limit, after which exopher production is suppressed. We grew mCherryAg2 animals to Ad1 at 20 C, and then, transferred animals to plates housing increasing concentrations of juglone, rotenone (rot), or paraquat (PQ) for 6 h, as indicated, measuring ALMR exophers shortly thereafter (black asterisks compare to control): 7 trials and 50 animals per trial. (G) Combined food withdrawal and osmotic stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on plates containing the solutes indicated (20 C) and scored for ALMR exophers shortly thereafter: 3 trials and 50 animals per trial (asterisks compare two indicated conditions at bar ends). (H) Combined food withdrawal with oxidative stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on juglone, rotenone, and paraquat at the concentrations indicated, scoring for ALMR exophers shortly thereafter: 5 trials and 50 animals per trial (asterisks compare two indicated conditions). (I) While anoxia does not increase exophers, combining food withdrawal and anoxic stress can suppress fasting-associated exopher induction. We fasted Ad1 mCherryAg2 animals for 6 h under anoxic conditions, scoring for ALMR exophers shortly thereafter: 4 trials and 50 animals per trial. Note that our anoxia protocol is not effective in limiting fasting-induced exopher elevation (SI Appendix, Fig. S2 D and H).

We also quantitated exopher levels under conditions of long-term, hyperosmotic stress during adult life (Fig. 2B). We raised animals under standard growth conditions until the L4 stage and then moved animals to 250 mM sucrose, glucose, sorbitol, or NaCl plates for adult life. We first measured exopher levels 24 h after initiating osmotic stress and tested the same population on Ad1 to Ad4 thereafter. For all solutes tested, osmotic stress resulted in an 5 increase in exophers over the baseline at peak and shifted the measured peak of exopher onset forward 1 d. Notably, exopher levels returned to baseline at Ad4, even in the presence of osmotic stressors. We conclude that both transient and extended exposure to hyperosmotic stress can elevate exopher production substantially in early adult life and infer that hyperosmotic conditions rapidly generate a trigger that elevates exophers.

Oxidative stress is a central player in aging biology and proteostasis (21). We tested whether well-characterized chemical inducers of mitochondrial oxidative stress can influence exopher production in ALMR neurons. We exposed Ad1 mCherryAg2 animals to 6 h of increasing concentrations of juglone (juglone is a plant-derived compound that can induce superoxide production when incorporated into nematode growth medium [NGM]); rotenone, which interacts with mitochondrial electron transport complex I to elevate reactive oxygen species (ROS); and paraquat, which increases mitochondrial superoxide production (Fig. 2 DF). For all mitochondrial ROS generators, we observed a dose-dependent increase in exophers at Ad1 (to a point, discussed in Excessive Stress Decreases Exopher Production) that ranged from 4 to 6 over untreated controls. We conclude that exposure to mitochondrial ROS induction can enhance exopher production.

We also tested conditions of continuous ROS exposure during adult life. We raised animals under standard growth conditions until the L4 stage and shifted cultures to paraquat plates, measuring ALMR exophers on Ad1 to Ad8 (SI Appendix, Fig. S2E). We find that continuous paraquat exposure increases exopher levels. Interestingly, exopher levels in the 2-mM paraquat treatment do not begin to fall at Ad3, as is typical for other stresses, but instead remain higher than control levels through Ad6, returning to baseline at Ad7. Thus, paraquat-mediated oxidative stress can extend the time period of permissive exophergenesis by 2 d, raising the possibility that paraquat might induce signals that normally promote exopher production. Overall, data from three mitochondrial ROS-generating compounds indicate that chemically-induced oxidative stress can increase exopher formation.

While initially testing the ability of stressors to modulate exopher production, we often utilized a doseresponse approach. Our studies revealed a striking commonality regardless of stressor type: excessive stress suppresses exophergenesis. For example, 6-h exposure of 240 M juglone (Fig. 2D), 25 M rotenone (Fig. 2E), or 25 mM paraquat (Fig. 2F) reduces exopher levels, even though lower levels of these ROS stressors enhance exopher production. The same pattern emerges under osmotic stress conditions; 6-h exposure to 500 mM concentrations of sucrose, glucose, sorbitol, and NaCl suppresses exopher levels, compared to 250 mM concentrations of each of these solutes (Fig. 2C). Although at Ad1 ALMR exophers modestly increase with 6-h exposures to increasing temperatures up to 30 C, 6 h at 37 C causes a collapse in exophergenesis (SI Appendix, Fig. S2F).

We generated additional evidence in support of the idea that excessive stress can inhibit exopher formation by exposing Ad1 mCherryAg2 animals to combined two-stress conditions that, by themselves, individually enhance exopher production. For example, whereas a 6-h fast elevates exophers (Figs. 1 BF and 2G), cointroducing osmotic stress with fasting (which also normally also elevates exophers; Fig. 2 A and B) suppresses exopher levels (Fig. 2G). We also found a combined inhibitory effect for fasting + oxidative stress (Fig. 2H).

Furthermore, temperature (SI Appendix, Fig. S2G) and anoxia (Fig. 2I and SI Appendix, Fig. S2H), stresses that did not affect exophergenesis on their own (SI Appendix, Fig. S2 A, B, and D), could suppress the effects of fasting on exopher production. Data suggest that under extreme stress neurons either cannot meet molecular requirements for exopher production or might enact mechanisms that actively suppress exophergenesis (see Discussion).

In summary, food withdrawal, osmotic stress, and oxidative stress can enhance exopher production, although above specific threshold levels of these stresses (including when two individual, exopher-promoting stresses are combined), exopher production can be suppressed. Thus, in addition to a temporal constraint on when environmental stresses can elevate exopher levels, we demonstrate that there is a limit to the severity of environmental stress capable of inducing the exopher production response.

With a goal of defining molecular mechanisms by which stresses elevate neuronal exopher production, we sought to define genetic requirements for the fasting-dependent induction response. We elected to focus on the fasting response because of the robust and highly reproducible level of induction associated with 6 h food withdrawal (Fig. 1 B and C), combining genetic mutant and RNAi strategies. To probe the mechanism by which fasting elevates neuronal exopher production, we first tested mutants for well-characterized, stress-activated transcription factors: heat shock factor 1 hsf-1/HSF1, required for transcription of heat shock chaperones and proteostasis (22); hypoxia inducible factor hif-1/HIF1, required for hypoxia stress responses (23); hlh-30/TFEB, required for starvation resistance and lysosomal integration with metabolism (24); skn-1/NRF2, which promotes response to oxidative stress and xenobiotic challenge (25, 26); and daf-16/FOXO, which is activated by low insulin pathway signaling and functions in a range of stress-protective responses, including proteostasis (27).

We constructed mCherryAg2 strains with viable mutant alleles of each transcription factor and subjected mutants to 6 h food deprivation on Ad1, measuring ALMR exophers thereafter (Fig. 3A). We observed the significant elevation of exophergenesis in hsf-1, hif-1, hlh-30, and skn-1 backgrounds in response to food withdrawal, indicating that these transcription factors are not critical for the induction of exophers in response to fasting. In contrast, the daf-16 null mutant exhibited a partial defect in the food withdrawal response, with exopher levels clearly increasing over baseline in the absence of food (P < 0.001) but never reaching the levels observed in wild-type (WT) animals (P < 0.001) (Fig. 3A). The partial effect in the daf-16 null mutant background is consistent with a model in which a daf-16dependent process mediates one component of the fasting response but that a daf-16independent process works in parallel to elevate exophers when food is withdrawn.

Lipid synthesis and FGF-activated RAS/ERK signaling are required for the induction of neuronal exophers in response to fasting. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. Feeding RNAi was initiated at the L4 larval stage and continued until Ad2. (A) The daf-16 null mutant has diminished exopher production in response to fasting. We tested mutants defective in major stress-responsive transcription factors (daf-16/FOXO, hsf-1/HSF-1, hif-1/HIF1, hlh-30/TFEB and skn-1/NRF2) in the mCherryA2 background, Ad2: exopher counts 6 trials, 50 animals per trial, and CMH difference between WT control and daf-16 deletion mutant ***P < 0.001. (B) Lipid synthesis is implicated in the fasting-induced boost in exophergenesis. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 beginning at L4 in the mCherryAg2 strain, 6 h fast followed by exopher assay on Ad2, control is empty vector RNAi. We compared WT fed versus fasted P < 0.001 CMH, the others are not significant: 3 trials and 50 animals per trial. (C) Schematic of C. elegans MAPK signaling pathways targeted in genetic tests for fasting-induced exopher elevation: p38 MAPK signaling (purple), JUN/FOS MAP kinase signaling (orange), EGF-mediated MAPK signaling (green), and FGF-mediated MAPK signaling (blue). Black boxes highlight a common function in both EGF and FGF MAPK signaling. (D) RNAi knockdown of most components of the p38 and JUN/FOS signaling cascades does not impair fasting-induced exopher increases. The strain was mCherryAg2; RNAi was initiated at the L4 stage; and 6 h fast was followed by exopher counts at Ad2: 3 trials and 50 animals per trial. mek-1(RNAi) stood out as exceptional in failing to induce exophers. (E) Loss-of-function mutants for JUN/FOS signaling do not suppress exopher production. kgb-1; mCherryAg2 and jun-1; mCherryAg2 mutants were fasted for 6 h at Ad2 before exopher counts: 5 trials and 50 animals per trial. (F) An egl-17/FGFmediated MAPK signaling cascade is necessary for a fasting-induced increase in exopher production. RNAi for the indicated genes was initiated at the L4 stage on strain mCherryAg2, and at Ad2, animals were fasted 6 h and then scored for exophers. RNAi knockdown of let-23, ksr-2, and FGF ligand let-756 did not disrupt the fasting exopher response 3 trials and 50 animals per trial.

We also tested whether autophagy, a pathway activated by food limitation, might be critical for the fasting-induced increase in exophers. We used RNAi approaches to knockdown autophagy genes lgg-1, atg-7, and bec-1 in mCherryAg2 animals and scored for exophers on Ad2 (SI Appendix, Fig. S3A). Since all three disruptions in the autophagy pathway failed to suppress fasting-induced exopher elevation, we infer that engagement of autophagy functions is not required for fasting-induced exopher increase. Pharmacological inhibition of autophagy with 1 mM spautin or of the proteasome with 10 mM MG132 (SI Appendix, Fig. S3B) did not block the fasting-induced elevation of exophers, in further support that autophagy and proteasome contributions are not critical for fasting-induced exopher increase.

To better characterize the pathways involved in fasting-induced exopher production, we compiled a list of known genes implicated in starvation and feeding in the C. elegans literature and screened for fasting-induced exopher elevation when the candidate genes were knocked down using RNAi (28) (see SI Appendix, Table S1 for a list of candidates tested). Note that the strain we targeted with feeding RNAi, mCherryAg2, should permit efficient RNAi knockdown in all tissues except neurons, because neurons do not express a double-stranded RNAi transporter, sid-1, required for the efficacious knockdown in feeding RNAi (29). Utilizing this experimental design, we expected to identify genes operative in the nonautonomous initial events in the sensation and signaling of fasting stress to the touch neurons, rather than genes involved in neuron-intrinsic exophergenesis.

We tested positive clones from the first round of the RNAi screen in triplicate to identify intestinal peptide transporter pept-1, lipid synthesis-implicated Mediator complex factor mdt-15, MDT-15 binding partner sbp-1/SREBF2, and fatty acid synthase fasn-1, as required for robust exopher elevation in response to fasting (Fig. 3B).

pept-1 encodes a conserved intestinal di-/triamino acid transporter implicated in C. elegans nutrient sensing (30). Loss of the pept-1 function increases the intestinal absorption of free fatty acids from ingested bacteria, such that short- and medium-chain fatty acids are highly increased in the mutant, and de novo synthesis of long-chain and polyunsaturated fatty acids is greatly decreased (31). Our data suggest that sudden withdrawal of food causes a metabolic reconfiguration that signals for enhanced exopher production and that the sensing of food limitation requires PEPT-1 transporter activity.

MDT-15 has been shown to promote health and longevity by orchestrating many of the metabolic changes that occur in response to short-term fasting (32). MDT-15 encodes a subunit of the transcriptional coregulator Mediator complex that is required to express fatty acid metabolism genes and fasting-induced transcripts (3235), heavy metal and xenobiotic detoxification genes (32, 36), and oxidative stress genes (37). SBP-1, the homolog of the mammalian sterol regulatory element-binding protein (SREBF2) transcription activator that regulates fatty acid homeostasis, is a known partner of MDT-15, and together, MDT-15 and SBP-1 promote the expression of lipid synthesis genes (32). FASN-1 encodes the sole C. elegans fatty acid synthase, and its expression is regulated by MDT-15/SBP-1 (38, 39). Together, MDT-15, SBP-1, and FASN-1 may act to promote the synthesis of a lipid-based factor that signals for, or is otherwise required for, neuronal exopher production under fasting stress.

Expression of lipid synthesis genes in multiple tissues may contribute to fasting-induced exopher elevation. To address where the lipid synthesis gene group required for fasting-induced exopher elevation acts, we took a tissue-specific RNAi knockdown approach. We worked with strains that expressed mCherryAg2 in touch neurons but were defective in either the double-stranded RNA (dsRNA) transporter sid-1 (neurons, muscle, pharynx, intestine, and hypodermis) or RISC complex factor rde-1 (germline and vulva), and we reintroduced sid-1 or rde-1 using tissue-specific promoters to drive expression and restore RNAi knockdown capability only in the rescued tissue. The promoters we used to restore expression (and therefore RNAi targeting) in specific tissues were the following: pan-neuronal rgef-1, muscle myo-3, pharynx myo-2, intestine vha-6, hypodermis hyp7 semo-1, vulva lin-31, and germline sun-1. This test set of seven tissue-specific RNAi lines enabled us to target most cells of the animal. We fasted animals to ask whether RNAi disruption in individual tissues is sufficient to disrupt the exopher induction response, which would indicate that expression in that targeted tissue is necessary in the response. We found that although whole body knockdown of pept-1, mdt-15, sbp-1, and fasn-1 could disrupt the fasting-induced exopher elevation (Fig. 3B), no tissue-specific knockdown was effective in blocking this response (SI Appendix, Fig. S4A). Data are consistent with a model in which multiple tissues can contribute the required lipid biosynthesis, although we cannot rule out that RNAi targeting was ineffective or missed necessary cells.

Food limitation stresses engage multiple signaling pathways that can activate animal defenses, including RAS/MAPK pathways that transduce developmental and stress responses to activate specific transcription programs (40, 41). We tested members of three canonical, well-characterized C. elegans MAPK signaling pathwaysthe PMK-1/p38 pathway that functions in some innate immunity and oxidative stress responses; the JNK pathway, which, among other activities, functions in intermittent fasting programs (42); and the RAS/ERK pathway, which, among other things, affects vulval precursor fate and vulval development response to starvation stress (43) (pathways summarized in Fig. 3C).

The core MAP kinases in the p38 pathway are nsy-1/MAPKKK and pmk-1/MAPK (40). RNAi directed against nsy-1 and pmk-1 genes was ineffective in blocking the fasting induction of neuronal exophers (Fig. 3D), and thus, we do not find evidence supporting the engagement of the pmk-1/p38 stress pathway in fasting-induced exopher elevation.

In C. elegans, an adult intermittent fasting protocol of 2 d without food followed by 2 d of food extends lifespan via an MLK-1, MEK-1, and KGB-1 JNK pathway that converges on AP-1 (JUN-1, FOS-1)mediated transcription (42). We find that kgb-1(RNAi), kgb-1, and jun-1 genetic mutations do not block the fasting-induced elevation of exopher production (Fig. 3 D and E). This result, coupled with published data examining a transcriptional time course following food withdrawal that revealed a distinct transcription pattern for 3 to 6 h after fasting, as compared to the more chronic starvation of 9 h and longer (44), suggests that the response to short-term food withdrawal that we characterize here does not operate via the characterized, intermittent fasting pathway. Still, the positive mek-1/MAPKK and partial mlk-1/MAPK outcome (mlk-1(RNAi) fasted versus WT fasted, not significant; mlk-1(RNAi) fasted versus mlk-1(RNAi) fed, P < 0.01) (Fig. 3D), suggest possible pathway involvement or cross-talk involving these kinases. As evidence for more definitive engagement of the RAS/ERK pathway was evident in our studies (Fig. 3F), we focused on examining pathway members in more detail.

RAS/ERK signaling in C. elegans (reviewed in refs. 40 and 41; see Fig. 3C) can involve the EGF or FGF activation of receptor tyrosine kinases that interact with adaptor proteins such as SEM-5/GRB2 or SOC-1/GAB1, which recruit guanine nucleotide exchange factor SOS-1 to activate small GTPase LET-60/RAS. LET-60/RAS-GTP activates MAPKKK LIN-45/RAF. Scaffold proteins KSR-1 and/or KSR-2 collect downstream members of the MAPK cascade, so LIN-45/MAPKKK activates MEK-2/MAPKK, which in turn phosphorylates and activates MPK-1/ERK. MPK-1 can then enter the nucleus to phosphorylate transcription factors that execute a transcriptional response. We found RNAi knockdown of multiple members of the conserved core RAS/ERK signaling, namely let-60/RAS, mek-2/MAPKK, mpk-1/ERK, sem-5, and ksr-1, disrupts the fasting-induced increase of touch neuron exophers (Fig. 3F). Given the implication of five core MAPK components in fasting-induced exopher elevation, we conclude that the RAS/ERK pathway plays a critical role in the mechanism by which neurons increase mCherry expulsion (and other cell contents) under food withdrawal stress.

The characterized C. elegans EGF and FGF signaling pathways share the aforementioned signaling components in the conserved RAS/ERK pathways but differ in ligands, receptors, KSR-type (EGFR uses both KSR-1 and KSR-2; FGFR exclusively uses KSR-1), and FGF pathway requirement for the adaptor SOC-1/Gab1 (41). We tested MAPK components in an effort to distinguish whether FGF, EGF, or both, contribute to exopher elevation in response to fasting.

We used RNAi approaches to test for a requirement of FGF ligands egl-17 and let-756 as well as FGF receptor egl-15 in fasting-induced exopher elevation. Interventions with ligand egl-17/FGF and receptor egl-15/FGFR, but not FGF ligand let-756, disrupted the capacity to increase exophers in response to 6-h fasting, implicating a specific FGF in exopher regulation biology (Fig. 3F). Moreover, we find that FGF pathway-specific soc-1 (RNAi) diminishes fasting-induced exopher production. Our data, which implicate eight FGF pathway genes (FGF ligand egl-17, FGF receptor egl-15, FGF pathway-specific soc-1 and sem-5, and core pathway components let-60, mek-2, ksr-1, and mpk-1) in the fasting induction of exophers, identify an FGF-activated ERK/MAPK pathway that acts in response to 6-h food withdrawal, as an essential mechanistic step in neuronal exopher increase.

To address the potential source of egl-17/FGF and identify the tissue via which FGFR acts to promote fasting-elevated exopher production, we used tissue-specific RNAi approaches to disrupt all identified components in all neurons, muscle, pharynx, intestine, hypodermis, vulva, or germline (SI Appendix, Fig. S4A).

We were unable to identify single-tissue sources that were required for FGF ligand EGL-17 or FGF receptor EGL-15 activity in fasting-induced exopher elevation, suggesting that either multiple tissues can execute effective FGF signaling, that cells that we were unable to target are responsible, or that unknown technical issues might apply. We did, however, find that knockdown of the most downstream pathway targets, mek-2 and mpk-1, only in the hypodermis or only in the germline (SI Appendix, Fig. S4B) could disrupt fasting induction. Our data thus suggest that mek-2 and mpk-1 are required in both the hypodermis and in the germline for fasting-induced exopher induction.

Importantly, mek-2 and mpk-1 are downstream kinases for both the FGF and the EGF MAPK pathways. Indeed, our RNAi perturbation screens of MAPK pathway members indicated that lin-3/EGF also acts in fasting-induced exopher elevation (Fig. 3F). Tissue-specific RNAi studies reveal that germline-specific knockdown of lin-3/EGF disrupted fasting-induced exopher elevation (while hypodermal knockdown did not) (SI Appendix, Fig. S4C), implicating the germline production of LIN-3/EGF in a transgenerational influence on neuronal exophergenesis. Knockdown of mek-2 and mpk-1 are also needed in the germline for fasting-induced exophergenesis (SI Appendix, Fig. S4B).

RNAi disruption of EGF receptor let-23 and EGF pathway-specific ksr-2 did not eliminate the exopher elevation in response to fasting in our original screen (Fig. 3F). This observation raised the possibility that the EGF-responsive pathway might function in neurons (which are generally not as susceptible to RNAi as other tissues). Because let-23(gf) alleles caused extensive reproductive system development consequences that complicated the interpretation of exopher production (not shown), we tested the available strains that expressed the EGF pathway-activating EGFR/let-23(gf) in specific tissues for elevated exopher levels in the presence of food. We found that the transgenic introduction of EGFR/let23(gf) in neurons (but not muscle or intestine) elevates exophers in the presence of abundant food (Fig. 4A), suggesting a neuronal-based EGF pathway in promoting fasting-induced exopher elevation.

Molecular pathways that influence fasting-induced exopher elevation. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) let-23 (gain of function) increases exopher formation when expressed specifically in neurons. let23(sa62) is a gain-of-function allele that activates MAPK signaling. We measured the exopher levels in strains that expressed let-23(sa62gf) from neuronal (unc-119), intestinal (vha-6), or muscle (myo-3) promoters in the mCherryAg2 background at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (B) let-60 (gain of function) increases exopher formation under abundant food conditions. let-60(n1046) is a gain-of-function RAS allele that activates MAPK signaling. We measured exopher levels in control mCherryAg2 and in let-60(n1046);mCherryAg2 strains at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (C) Schematic of the logic of epistasis within the MAPK signaling context. The phenotype of a gain-of-function allele that continuously signals will not be affected by the knockdown of any effector upstream of it within the cascade. On the other hand, any actors downstream of a constitutively active component will affect the phenotype. FGF signaling is used as an example here. (D) Lipid synthesis genes act upstream of let-60(gf)/RAS to elevate neuronal exopher production. RNAi interventions were initiated at the L4 stage on gain-of-function let-60;mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Although mek-2 and mpk-1, known to act downstream of let-60(gf), are critical for exopher elevation, the known upstream genes egl-15, egl-17, and soc-1 are not, consistent with data that previously ordered the FGF pathway. pept-1, mdt-15, sbp-1, and fasn-1 do not suppress let-60(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of let-60/RAS. (E) Lipid synthesis genes and lin-3 act upstream of let-23(gf) activity when it is expressed in neurons. RNAi knockdown was initiated at the L4 developmental stage on gain-of-function let-23(sa62);mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Cascade components let-60, mek-2, and mpk-1 are known to act downstream of let-23 and effectively abrogate the MAPK signaling leading to exophers, while lin-3 works upstream of let-23 and therefore cannot suppress a downstream gain-of-function mutation. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 do not suppress neuronal let-23(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of neuronal let-23(gf). (F) Model for signaling that elevates neuronal exopher formation in response to fasting. In the sudden absence of food or when dipeptide transporter PEPT-1 activity is low, a lipid-based signal is produced/released that depends upon MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1. The lipid signal may act on the neurons directly or may contribute to the activation of a required FGF pathway in a relay tissue (unlikely to be neurons) to promote transcription of an essential signal that acts on touch neurons to elevate exopher production. EGF pathway activation in neurons can elevate exopher production; it remains to be determined whether EGF signaling is required in touch neurons per se. In sum, transtissue stress signaling influences remote neuronal trash management through lipid, FGF, and EGF RAS/MAPK signaling. Note that we cannot distinguish between an inducible lipid signal and a static, essential lipid product required for exopher elevation.

let-60(n1046) is a well-characterized, gain-of-function RAS allele that constitutively activates MAPK signaling (45). Our RNAi data, identifying the FGF and EGF/RAS/MAPK pathways as required for fasting-induced exopher induction, predict that the let-60(gf) allele should induce higher exopher levels, even in the absence of starvation. To test this model, we constructed a let-60(n1046gf);mCherryAg2 strain and measured exopher levels at Ad2. We find that exophers are indeed elevated in the let-60(n1046gf) background in the absence of fasting, confirming that RAS activation enhances exopher production (Fig. 4B).

let-60(n1046gf) has been used extensively in epistasis pathway ordering (45), and thus we pursued an epistasis approach to clarify the relationship between the lipid synthesis gene group and the FGF/RAS/MAPK pathways (Fig. 4C). We reasoned that if the genes involved in lipid synthesis act downstream or in parallel to the RAS/MAPK pathway that elevates exophers, RNAi knockdown of the lipid synthesis genes in the let-60(gf) background should suppress constitutive exopher production. Alternatively, if the lipid synthesis branch acts upstream of LET-60/RAS activation, disruption of the upstream genes should not change the exopher elevation associated with let-60(gf) (Fig. 4C). We therefore repeated RNAi knockdowns of genes in the lipid synthesis and FGF/RAS/MAPK pathways in the let-60(gf);mCherryAg2 background, quantitating the impact on exopher formation. As expected for known downstream kinases in the RAS pathway, the RNAi disruption of mek-2/MAPKK and mpk-1/MAPK suppressed the let-60(gf) phenotype (Fig. 4D). In contrast, genes encoding egl-17/FGF, egl-15/FGFR, and soc-1 that act upstream of let-60/RAS did not suppress the let-60(gf)/RAS phenotype, consistent with the known action of these genes upstream of RAS in the signaling pathway.

Importantly, RNAi directed against pept-1, and the lipid biosynthesis genes mdt-15, sbp-1, and fasn-1 did not suppress elevated exopher levels in the let-60(gf) background (Fig. 4D). We conclude that the lipid biosynthesis branch is likely to act upstream of the FGF/RAS/MAPK to promote exopher elevation.

Lipid and FGF signaling may act upstream of EGF signaling. We also used an epistasis strategy to begin to address how lipid biosynthesis and FGF pathways might relate to the EGF pathway in fasting-induced exopher elevation. We performed the RNAi knockdown of required lipid synthesis genes and of upstream FGF pathway-specific genes egl-17/FGF, egl-15/FGFR, and soc-1 on the neuronal let-23(gf) strain in which exopher levels are elevated, reasoning that if a critical lipid or FGF pathway is normally activated downstream of neuronal EGFR activation, perturbation of lipid or FGF pathway-specific genes would block the let-23(gf) exopher elevation under well-fed conditions. Our RNAi-dependent disruptions of either pathway, however, did not suppress neuronal let-23(gf) high-exopher levels (Fig. 4E), suggesting that the essential lipid and FGF signaling required for fasting-induced exopher induction normally occurs upstream of neuronal EGF signaling. Although these studies constitute only the first rudimentary tests required to establish pathway details (caveats discussed in more detail in SI Appendix, Fig. S4), data suggest a basic framework for mechanistic evaluation (Fig. 4F).

Overall, we identify three pathways that are required for fasting-induced exopher elevation in stressed touch receptor neurons: lipid synthesis, an FGF/MAPK pathway, and an EGF/MAPK pathway that can act in neurons. Our data suggest a model for signaling that influences a dramatic expulsion of neuronal contents upon the introduction of fasting stress. Upon food withdrawal, di-/tripeptide transporter PEPT-1 plays a role in nutrient sensing and a lipid-based stress signal (the generation of which depends on MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1) is produced. The lipid-dependent process could act directly to nonautonomously influence exopher production in the touch neurons or could trigger/activate the required FGF/RAS/MAPK and/or EGF MAPK signaling and the likely consequent downstream transcription. Downstream, or in parallel, RAS/MAPK activity in hypodermis and germline contributes to the stress-sensing tissue network, establishing that that nonautonomous signaling directs exopher production in the touch neurons. FGFR signaling pathway genes are known to be highly expressed in the hypodermis (46). Notably, RNAi evidence supports the hypothesis that the same group of genes that mediate fasting-induced exopher increase is required for exopher elevation in response to osmotic stress (SI Appendix, Fig. S3C), indicating that general, rather than fasting-specific, mechanisms are engaged.

Although many details of this complex lipid-FGF-EGF signaling network remain to be further elucidated, our data provide documentation that aging and proteostasis-relevant stresses engage multiple pathways that can act over multiple tissues to influence a dramatic expulsion of neuronal contents. Conserved signaling molecules can modulate a process of fundamental interest in neuronal proteostasis, relevant to the understanding of neuronal degeneration.

Maintaining neuronal proteostasis is a critical goal for healthy brain aging and a fundamental challenge for diseased neurons in a range of neurodegenerative diseases (47). A recently identified facet of Alzheimers disease, Parkinsons disease, and other proteopathies is the transfer of aggregates to neighboring cells, which can seed aggregate spread and promote pathology (48, 49). In vivo dissection of the biology of protein aggregate spread is challenging to investigate in mammalian brain, but it is clear that the understanding of mechanisms that regulate autonomous and nonautonomous aggregate expulsion in relation to other neuroprotective strategies is of considerable importance in addressing potential treatment.

C. elegans touch neuron exopher production, which increases with high proteostress (4), models several aspects of aggregate/organelle transfer biology. We find that the production of neuronal exophers can be dramatically responsive to specific stress conditions, being enhanced by food withdrawal, oxidative stress, and osmotic stress but influenced relatively little by temperature or hypoxia. We also demonstrate the temporal restriction of stress-induced exopher production to the first 3 d of adult life, and we document a stress ceiling phenomenon, in which the highest levels of individual stress, or a combination of two distinct noninhibitory stresses, suspend exopher production. Finally, we show that fasting-induced exophergenesis is dependent on nonautonomous lipid biosynthesis, FGF-activated RAS/MAPK, and EGF-activated RAS/MAPK signaling pathways. Although details remain to be filled in regarding the complex interactions of the signaling steps, a major point is that environmental and genetic factors can be manipulated nonautonomously to regulate the expulsion of offensive aggregates from neurons. Given the importance of aggregate management in aging and neurodegenerative disease and the poorly understood biology of in vivo aggregate transfer, exopher-related mechanisms may suggest new strategies toward the manipulation of the analogous process in higher organisms.

Acute food withdrawal, oxidative stress, and hyperosmotic stress elevate exopher production, but temperature elevation and hypoxia/anoxia are relatively ineffective at provoking similar responses. Starvation (8, 50), oxidative stress (51), and osmotic stress (52) perturb proteostasis and share ROS elevation (53, 54). The future dissection of the intersection of the genetic and physiological conditions common to these three stresses should provide insight into molecular mechanisms that promote exophergenesis. Likewise, physiological differences in stress responses to temperature and hypoxia, which are not potent inducers of exophers, may help distinguish particular conditions that are specifically correlated with exopher induction.

Exopher production, comprising the release of a large, membrane-surrounded vesicle filled with cellular contents, has the appearance of an energetically costly incident that involves the dynamic loss of organelles and aggregates. Our working model posits that exophergenesis is invoked when the levels of damaged organelles and proteins surpass the neuronal capacity for internal degradation. Consistent with this idea, increasing oxidative challenge and increasing hyperosmotic exposure both increase exophergenesis. Interestingly, conditions of extreme osmotic and oxidative stress markedly suppress the formation of exophers. Moreover, combining two stresses, either of which is sufficient to promote exopher production when introduced alone, can result in exopher suppression. This combinatorial effect can also occur with stress stimuli that themselves do not significantly induce exophers, such as anoxia and modestly elevated temperature. Together, these observations reveal a molecular summing of stress signals that appear to flip the off switch for exophergenesis. The suppression of exopher production under conditions of extreme stress may be caused by energy exhaustion, a molecular repression mechanism, or grievous loss of homeostasis, leading to physiological dysregulation.

Exopher production follows a distinctive and reproducible temporal profile in early adult life (4, 11). In the Ag2mCherry strain, exophers are not produced in larval development but begin to be detected after animals reach reproductive maturity, typically peaking in numbers around Ad2 and returning to low-baseline detection by Ad4. Data included here underscore that the temporal pattern is generally maintained, despite the continued or introduced presence of stresses. In other words, stresses definitively elevate exopher production, but for the most part, these stresses do not extend the period of exopher production later into adult life. Our findings thus define a limited temporal window in which exopher production can be modulated by stresses and suggest the existence of physiological states permissive (or restrictive) for exopher production. A link to reproduction likely defines this permissive period. For some stimuli (paraquat, rotenone, osmotic stress, and a shift to 25 C), we do report a capacity to move the peak day of exopher production ahead to Ad1 or at least to markedly enhance Ad1 levels above nonstressed controls. We infer that these stimuli reach the molecular threshold for exopher triggering faster than other conditions.

The fasting-induced elevation of exopher levels does not require stress transcription factors HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 but does depend in part on DAF-16/FOXO, a conserved stress-responsive transcription factor that drives the expression of food-sensitive, oxidative stress resistance and proteostasis genes (27, 44, 55) and is known to exert autonomous and nonautonomous impacts on stress resistance and longevity (56). Our data implicate a FOXO family member in regulation of neuronal aggregate expulsion. Our data do not rule out whether HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 might function redundantly in the exopher response to fasting.

How DAF-16 interacts with fat biosynthesis pathways and RAS/ERK signaling in exopher induction remains to be clarified. DAF-16 can control the expression of mdt-15 and has been previously implicated in transtissue benefits by interactions with MDT-15 (57). DAF-16 also intersects with FGF, ERK, and lipid biogenesis pathways and vice versa (for example, refs. 5861). The future definition of how DAF-16 integrates with these signaling pathways and the identification of the transcription factor that mediates the DAF-16independent component of the fasting-induced exopher response will add molecular understanding to what appears to be a complex regulatory network.

Mediator complex subunit MDT-15 is a transcriptional coregulator involved in lipid metabolism (34), response to fasting (32, 62), and oxidative, stress-induced expression of detoxification genes associated with the exposure to reagents like paraquat (63). The requirement for mdt-15 in fasting-induced, neuronal exophergenesis adds a new facet to the MDT-15 integration of multiple, transcriptional regulatory pathways (32), expanding the known roles of MDT-15/SBP-1 to include the activation of extrusion of remote neuronal aggregates in exophers. SBP-1/SREBF2 acts with MDT-15 to promote the expression of lipid metabolism genes (33, 34), and fatty acid synthase fasn-1 can be regulated by these (38, 39, 62, 64, 65). The requirement for multiple genes involved in lipid synthesis in fasting-induced exopher increase suggests that a lipid-based signal may be issued to ultimately direct or modulate neuronal trash expulsion. An equally plausible model is that lipid-dependent machinery is required for upstream signaling that promotes exophergenesis.

Our findings identify FGF ligand egl-17 (but not FGF ligand let-756), FGF receptor egl-15, FGF pathway specific soc-1, and pathway components let-60, mek-2, ksr-1, and mpk-1 as required for the fasting induction of exophers. These data reveal a specific FGF/RAS/ERK signaling pathway that enhances neuronal exopher production when food is withdrawn. Since the candidate RNAi screen for the factors required for fasting-induced exopher elevation was conducted in a strain background that is not readily permissive for neuronal RNAi effects and since RNAi knockdown of pathway components (specifically in neurons) does not block fasting-induced exopher increase, FGF/ERK signaling likely takes place outside of the touch receptor neurons to exert a regulatory role on neuronal exopher production. An interesting potential site of FGFR action is the hypodermis, which is necessary for some MEK-2/MPK-1 MAPK signaling. Such signaling is permissive for fasting-induced exopher production and the hypodermis is a known site of expression of FGFR pathway components (SI Appendix, Fig. S4B). Detailed, cell-specific expression studies will be required to test this model.

The conserved FGF pathway executes numerous roles in mammalian development and homeostasis (66). Interestingly, mammalian FGF21 acts as a global starvation signal that, among other things, impacts lipid metabolism. Although most FGF21 studies feature extended starvation and mousehuman differences have been noted (67), FGF21 is one of the most up-regulated rat liver genes under the conditions of an 8-h fast (68). FGF21 can cross the bloodbrain barrier to change hypothalamic neuron gene expression (69). Overall, the implication of FGF/ERK pathways in the response of neurons to food limitation across diverse metazoans suggests a mechanism that may be ancient and raises the possibility that the FGF branch of these pathways might activate extracellular trash expulsion mechanisms within mammalian neurons. If so, FGF signaling might be considered as a target for the therapeutic elimination of stored neuronal aggregates.

EGF RAS/MAPK signaling is also engaged in fasting-induced exopher elevation. The knockdown of the sole C. elegans EGF ligand lin-3 in whole animal, or only in germline, impaired fasting-induced exopher elevation.

Although RNAi knockdown of the EGFR let-23 and EGF pathway-specific ksr-2 in whole animal or only neurons was not effective, expressing activated, gain-of-function EGFR receptor allele let-23(sa62) in neurons resulted in elevated exopher levels in the absence of fasting. Epistasis studies suggest that EGFR activation in neurons could occur as a downstream target of lipid biosynthesis and FGF signaling. Although definitive establishment of fasting-induced EGFR activation in neurons remains (experimental caveats in interpretation of data are discussed in detail in SI Appendix, Fig. S4C), data are consistent with a role for EGF signaling originating in the germline as an inducer of EGFR-activated responses in neurons that promote exopher production. Future studies will need to confirm neuron requirements and to address whether EGFR directly activates exophergenesis in touch neurons or engages additional neurons as intermediatory signaling centers.

It is important that the germline serves as an EGF source needed for fasting-induced exopher elevation. Indeed, in studies of exopher production under standard growth conditions, we have defined a role for germline in the production of young adult exophers. A key point here is that food-sensing, nonautonomous growth factor signaling across generations can influence seemingly extreme neuronal proteostasis activity.

EGF- and FGF-dependent processes cooperate in development. For example, EGF signaling activates FGF production and a downstream FGF pathway required in vulval epithelial fate specification (70). Starvation conditions can influence the signaling level for the EGF/RAS/ERK pathway that specifies C. elegans vulval cell fateseither starvation or pept-1(RNAi)can enhance RAS/MAPK signaling during vulval fate specification (43). Our documentation of FGF signaling in food limitation responses that elevate neuronal proteostasis outcomes identifies a second C. elegans EGF- and FGF-regulated signaling pathway that responds to food limitation. Why food limitation might induce neuronal trash elimination is unclear. One possibility is that exophergenesis [which we track in single neurons in our study but is likely to also occur in other neurons and cells (4)] might serve as a mechanism to discard superfluous, neuronal proteins and organelles for degradative recycling in neighboring cells as resources become limited.

Our study defines the basic framework by which metabolic stresses engage a distributed network that influences a significant neuronal expulsion phenomenon. Evidence is accumulating that exopher-like extrusion capabilities are not limited to stressed C. elegans neurons [mammalian examples in refs. 71 and 72)]. For example, a recent comprehensive study of mitochondrial expulsion by mouse cardiomyocytes revealed numerous analogies between C. elegans exophers and mouse mitochondrial expulsion models (72). Although elaborating details of molecular homologies remain for the future, that related biology is likely to be conserved holds significant implications of interest with regard to mammalian aging and neurodegeneration. 1) It is interesting that stresses, particularly associated with aging (i.e., oxidative stress) or proteostasis impairment (i.e., osmotic stress), are especially potent in inducing C. elegans exopher elevation; the disruption of exopher-related biology may contribute generally to the decline/dysfunction in aging neurons across phyla. 2) Likewise, the direct demonstration that extreme stress levels, or the summation of distinct, nonconsequential stresses, can effectively shut down the exopher response suggests a type of potential excessive stress impairment relevant to pathological mechanisms. 3) Our data establish that exopher production can be responsive to specific, conserved biochemical signaling, such that chemical strategies for inducing, or limiting, the expulsion of neurotoxic material by exploiting exopher-related mechanisms in mammals might be considered targets for therapeutic manipulation.

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