eK0n0mi taK seriU$ d/h ekonomitakserius@blogspot.com

Juni 29, 2016

diam2suka: KETIDAKPAST1AN, ekh, gw mah hepi AJA : 010913_290616

Filed under: GLOBAL ECONOMY — bumi2009fans @ 12:54 pm

per tgl 29 Juni 2016, kondisi ihsg maseh dalam era POST BREXIT n IMPLEMENTASI @ tax amnesty 2016:

BLOOMBERG menyatakan tren NAEK @ ihsg, kok, sama sperti analisis sederhana gw

big-dancing-banana-smiley-emoticon

2013: 

Will volatility pop as traders return from beach? Adam Shell , USA TODAY 7 a.m. EDT September 1, 2013 Traders are returning from their Hamptons’ getaways and that could be a recipe for a market volatility pop. usa today NEW YORK — The Summer of ’13 was an eventful one on Wall Street. The stock market hit a record closing high on Aug. 2, followed by wild price gyrations blamed on worries about the Federal Reserve’s next policy move, geopolitical risk in Egypt and Syria, and a software glitch that shut down trading on the Nasdaq stock market one day for more than three hours. Thinking calm lies ahead might be a mistake. If the past is any guide, investors might want to buckle up for a wild ride when Wall Street traders return to work from their beach getaways in the Hamptons and elsewhere. The reason: September is the one month the Dow Jones industrial average has posted negative returns the past 100-, 50- and 20-year periods. And the ninth month of the year has caused wild price swings of 2% or more for the Dow’s monthly finish the past seven years. “Everyone is getting fearful over the dreaded month of September,” Ryan Detrick, senior technical strategist at Schaeffer’s Investment Research said in a blog post. There are a few things on the docket in September that has Wall Street on edge and could reinforce the month’s reputation for unpleasant volatility. Consider: • The Fed meeting mid-month could mark the start of its long-awaited “taper” of its massive monthly bond buying program. • Syria, and potential U.S. military involvement, remains a wild card. • The budget fight on Capitol Hill that could cause a government shutdown or worse. “All of these things combined create a sense of uncertainty,” says Andres Garcia-Amaya, global markets strategist at JP Morgan Funds. In 2012, the benchmark Dow, powered by what was then the Fed’s seemingly unending bond-buying stimulus, jumped 2.7%. In 2011, the blue-chip stock gauge plunged 6.2% amid fears of a Greece government bond default. In 2010, the Dow rose seven of the first trading days in September on its way to a 7.7% gain — a rally fueled by a slew of better-than-expected economic data. And way back in 2009, when the bull market was just six months old, the Dow rallied 2.3% after a key manufacturing index posted its first “growth” reading since January 2008 — signaling the economy’s return to recovery. September of 2008 was an especially infamous month, as Wall Street cratered under the weight of the bankruptcy filing of one-time titan Lehman Brothers — a shock to financial markets that knocked the Dow down 6%. But that scary drop followed on the heels of two back-to-back September gains for the Dow of 4% and 2.6% in 2007 and 2006, amid an earlier bull market fueled by low interest rates. Despite the recent rollercoaster rides on Wall Street and fresh threats to stocks, Garcia-Amaya advises investors with long-term investment horizons not to react. One might wrongly think a potential spike in volatility could be avoided by running to the safety of cash. “All of these uncertainties make you jittery, but deciding to go to all cash isn’t as safe as it sounds because cash is yielding 0%,” he says. “And you need a diversified portfolio to ensure that you don’t outlive your money.”

… kebayang ga, jika SUKU BUNGA DEPOSITO DI BANK = 0%, well, pasti BANK2 di INDONESIA SEPI NASABAH … tapi ITU YANG TERJADI DI AMRIK, DAN JEPANG, sementara di EUROZONE: 1% aja … maksudnya seh bagus, yaitu MENDUKUNG EKONOMI NEGARA2 TERSEBUT supaya BISA BERTUMBUH … tapi, yah, gitu lah, mosok cuma 0% atawa 1%, khan investor SAMA AJA BOONG DONG NYIMPAN DI BANK … SERBA DILEMATIS seh, tapi itu lah PASTI DI DUNIA INI YAITU KETIDAKPASTIAN … sila baca posting gw soal ketidakpastian: justru KETIDAKPASTIAN ITU merupakan KEPASTIAN BWAT MASA DEPAN GW YANG LEBE BAEK JANGKA PANJANG BERINVESTASI gw BERLABA seh, beneran JANGKA PANJANG @ saham, reksa dana saham, n emas … mending

CARI AMAN AJA dan HIDUP DALAM KEPASTIAN YANG MEMBERIKAN KETIDAKPASTIAN MASA DEPAN

… sila pilih sendiri ya, tanggungjawab sendiri lah … 🙂 … he3, neh baca lage, supaya MAKIN CEMAS YAAAAAAAAA : August 31, 2013, 8:14 p.m. ET Markets May Be In for Rocky Autumn Fed action, Syria and wrangling over the budget and debt ceiling will weigh on investors By GREGORY ZUCKERMAN CONNECT As investors return from summer holidays to reconsider their portfolios, they’re facing two big questions: Has the market’s recent selloff been enough to make stocks attractive? And can the market rouse itself for a fall rally, even as the Middle East turns volatile and the Federal Reserve prepares to reduce the help it has been giving the economy? U.S. stocks and bonds rose for much of the year, but all kinds of investments ran into recent trouble during the summer. After reaching a high for the year on Aug. 2, the S&P 500 has fallen 4.5%, while the Dow Jones Industrial Average is off 5.4%. The Nasdaq Composite Index is down 2.8% from its 52-week high hit Aug. 5. Foreign markets have done even worse, especially emerging-market nations such as India, Brazil, Indonesia and Turkey. The Dow Jones Total Stock Market Emerging Markets Index has fallen over 6% since Aug. 2. Bonds also have struggled. After dropping to 1.6% in early May, the yield on the benchmark 10-year Treasury note (which moves in the opposite direction to its price) rose to 2.84% recently, one of the sharpest moves in years. On Friday, the 10-year note’s yield was at 2.75%. Behind all the troubles is a growing consensus that the economy has stabilized enough to allow the Fed to “taper” its easy-money program, highlighted by the $85 billion in monthly bond purchases. Some say this will come as soon as this month. That’s discouraging because at least some of this year’s gains are due to the Fed’s help, rather than improving fundamentals in the economy, analysts say. Most economists expect the U.S. economy to grow at an unremarkable 3% or so next year, and earnings expectations have been falling. If the Fed isn’t around to help prop up the market and economy, there will be less reason to get excited about stocks, some caution. “Now that it’s widely acknowledged that Daddy’s taking the T-Bird away,” investors will be forced to look critically at earnings and revenues as a source of support, says Jack Ablin, chief investment officer of BMO Private Bank, adding that much of this year’s rally has been fueled by the Fed’s monthly cash infusion. Another reason to be hesitant before buying U.S. stocks: September has been the worst month for stock performance. Going back to 1928, the market has been down 1.1% on average in the month, according to Merrill Lynch—making it by far the worst month of the year, though analysts have few obvious reasons for the underperformance. Meanwhile, oil prices have climbed, as the conflict in Syria has heated up, which could put pressure on consumer spending. The market also could be affected by the need for a budget resolution in Washington as the fiscal year comes to an end, and more debt-ceiling negotiations. There’s reason to be more cautious about foreign markets than the U.S., however, since the U.S. economy likely will prove more resilient. While the Fed is expected to slow its bond buying, there’s no reason to anticipate a move to raise interest rates any time soon. A big part of the reason the Fed will pull back is because near-term expectations for the federal deficit are improving. Gary Evans, a former trader and author of the Global Macro Monitor blog, notes that investors could shift cash to U.S. markets from abroad, as the emerging-market pain continues. Kathleen Kelley, who runs hedge fund Queen Anne’s Gate Capital Management in New York, says “the next few months will show Europe stagnating again and China stabilizing around these levels. However, U.S. growth will continue to pick up through year-end.” An increase in 10-year Treasury rates simply returns them to a normalized range following a period of historic lows in the aftermath of the deepest economic downturn since the Great Depression. The rise isn’t expected to be enough to undermine the housing market’s impressive recovery, most analysts say. The rate for a 30-year, fixed-rate mortgage stands at 4.58%, up from 3.59% two months ago. At the same time, stocks have become more attractive on the heels of their summertime weakness. The S&P 500 is trading at a price/earnings multiple of less than 14 based on expected earnings this year. That’s not a blaring buy signal, analysts say, but it suggests the stock market isn’t trading at especially expensive levels—another reason investors should hesitate before trimming too much from their portfolios. Meanwhile, oil prices could drop back as new supplies from Libya and elsewhere come to the market, some analysts say. And while September is a historically poor month for stocks, the last three months of the year have usually been good ones. Merrill Lynch strategist Savita Subramaian says technology, industrial and energy stocks will be the market’s leaders in the latter part of 2013 and early 2014. The firm recommends investors resist the urge to shift back to bonds, despite higher interest rates, and stick with stocks, arguing that stocks now are fairly valued, though not at bargain levels. Andrew Milligan, head of global strategy for Standard Life Investments, believes that markets will remain turbulent, with the Syrian conflict and moves by global central banks taking center stage during the year’s final months. But the market should hold up because he doesn’t expect banks to make “major policy errors.” … bayangin aja 2 HARIAN UTAMA amrik lho, ngoceh NEGATIF BANGET ya … kayanya kiamat neh, kiamat keuangan seh … 🙂 … well, bandingkan dengan posting berikut ini : KRUGMAN versus gw, mana yang lebe BERBOBOT analisis n prediksinya … 🙂 August 29, 2013 The Unsaved World By PAUL KRUGMAN new york times The rupiah is falling! Head for the hills! On second thought, keep calm and carry on. In case you’re wondering, the rupiah is the national currency of Indonesia, and, like many other emerging-market currencies, it has fallen a lot over the past few months. The thing is, the last big rupiah plunge was in 1997-98, when Indonesia was the epicenter of an Asian financial crisis. In retrospect, that crisis was a sort of dress rehearsal for the much bigger crisis that engulfed the advanced world a decade later. So should we be terrified about Asia all over again? I don’t think so, for reasons I’ll explain in a minute. But current events do bring back memories — and they are, in particular, a reminder of how little we learned from that crisis 16 years ago. We didn’t reform the financial industry — on the contrary, deregulation went full speed ahead. Nor did we learn the right lessons about how to respond when crisis strikes. In fact, not only have we been making many of the same mistakes this time around, in important ways we’re actually doing much worse now than we did then. Some background: The run-up to the Asian crisis bore a close family resemblance to the run-up to the crisis now afflicting Greece, Spain and other European countries. In both cases, the origins of the crisis lay in excessive private-sector optimism, with huge inflows of foreign lending going mainly to the private sector. In both cases, optimism turned to pessimism with startling speed, precipitating crisis. Unlike Greece et al., however, the crisis countries of 1997 had their own currencies, which proceeded to drop sharply against the dollar. At first, these currency declines caused acute economic distress. In Indonesia, for example, many businesses had large dollar debts, so when the rupiah plunged against the dollar, those debts ballooned relative to assets and income. The result was a severe economic contraction, on a scale not seen since the Great Depression. Fortunately, the bad times didn’t last all that long. The very weakness of these countries’ currencies made their exports highly competitive, and soon all of them — even Indonesia, which was hit worst — were experiencing strong export-led recoveries. Still, the crisis should have been seen as an object lesson in the instability of a deregulated financial system. Instead, Asia’s recovery led to an excessive showing of self-congratulation on the part of Western officials, exemplified by the famous 1999 Time magazine cover — showing Alan Greenspan, then the Fed chairman; Robert Rubin, then the Treasury secretary; and Lawrence Summers, then the deputy Treasury secretary — with the headline “The Committee to Save the World.” The message was, don’t worry, we’ve got these things under control. Eight years later, we learned just how misplaced that confidence was. Indeed, as I mentioned, we’re actually doing much worse this time around. Consider, for example, the worst-case nation during each crisis: Indonesia then, Greece now. Indonesia’s slump, which saw the economy contract 13 percent in 1998, was a terrible thing. But a solid recovery was under way by 2000. By 2003, Indonesia’s economy had passed its precrisis peak; as of last year, it was 72 percent larger than it was in 1997. Now compare this with Greece, where output is down more than 20 percent since 2007 and is still falling fast. Nobody knows when recovery will begin, and my guess is that few observers expect to see the Greek economy recover to precrisis levels this decade. Why are things so much worse this time? One answer is that Indonesia had its own currency, and the slide in the rupiah was, eventually, a very good thing. Meanwhile, Greece is trapped in the euro. In addition, however, policy makers were more flexible in the ’90s than they are today. The International Monetary Fund initially demanded tough austerity policies in Asia, but it soon reversed course. This time, the demands placed on Greece and other debtors have been relentlessly harsh, and the more austerity fails, the more bloodletting is demanded. So, is Asia next? Probably not. Indonesia has a much lower level of foreign debt relative to income now than it did in the 1990s. India, which also has a sliding currency that worries many observers, has even lower debt. So a repetition of the ’90s crisis, let alone a Greek-style never-ending crisis,

seems unlikely.

What about China? Well, as I recently explained, I’m very worried, but for entirely different reasons, mostly unrelated to events in the rest of the world. But let’s be clear: Even if we are spared the spectacle of yet another region plunged into depression, the fact remains that the people who congratulated themselves for saving the world in 1999 were actually setting the world up for a far worse crisis, just a few years later.

So should we be terrified about Asia all over again?I don’t think so

Juni 26, 2016

beautiful: robot dna, genom BUATAN V kanker manusia … 170212_270616

Filed under: Medicine — bumi2009fans @ 12:35 am

 

Pandangan SEDERHANA gw @ Brexhit, ooops, BRESHIT, ooops: BREX1T

long jump icon

the easy way to edit dna

SPINY GRASS AND SCRAGGLY PINES creep amid the arts-and-crafts buildings of the Asilomar Conference Grounds, 100 acres of dune where California’s Monterey Peninsula hammerheads into the Pacific. It’s a rugged landscape, designed to inspire people to contemplate their evolving place on Earth. So it was natural that 140 scientists gathered here in 1975 for an unprecedented conference.

They were worried about what people called “recombinant DNA,” the manipulation of the source code of life. It had been just 22 years since James Watson, Francis Crick, and Rosalind Franklin described what DNA was—deoxyribonucleic acid, four different structures called bases stuck to a backbone of sugar and phosphate, in sequences thousands of bases long. DNA is what genes are made of, and genes are the basis of heredity.

Preeminent genetic researchers like David Baltimore, then at MIT, went to Asilomar to grapple with the implications of being able to decrypt and reorder genes. It was a God-like power—to plug genes from one living thing into another. Used wisely, it had the potential to save millions of lives. But the scientists also knew their creations might slip out of their control. They wanted to consider what ought to be off-limits.

By 1975, other fields of science—like physics—were subject to broad restrictions. Hardly anyone was allowed to work on atomic bombs, say. But biology was different. Biologists still let the winding road of research guide their steps. On occasion, regulatory bodies had acted retrospectively—after Nuremberg, Tuskegee, and the human radiation experiments, external enforcement entities had told biologists they weren’t allowed to do that bad thing again. Asilomar, though, was about establishing prospective guidelines, a remarkably open and forward-thinking move.

Subscribe to WIRED PHOTO BY:RICHARD MOSSE

At the end of the meeting, Baltimore and four other molecular biologists stayed up all night writing a consensus statement. They laid out ways to isolate potentially dangerous experiments and determined that cloning or otherwise messing with dangerous pathogens should be off-limits. A few attendees fretted about the idea of modifications of the human “germ line”—changes that would be passed on from one generation to the next—but most thought that was so far off as to be unrealistic. Engineering microbes was hard enough. The rules the Asilomar scientists hoped biology would follow didn’t look much further ahead than ideas and proposals already on their desks.

Earlier this year, Baltimore joined 17 other researchers for another California conference, this one at the Carneros Inn in Napa Valley. “It was a feeling of déjà vu,” Baltimore says. There he was again, gathered with some of the smartest scientists on earth to talk about the implications of genome engineering.

The stakes, however, have changed. Everyone at the Napa meeting had access to a gene-editing technique called Crispr-Cas9. The first term is an acronym for “clustered regularly interspaced short palindromic repeats,” a description of the genetic basis of the method; Cas9 is the name of a protein that makes it work. Technical details aside, Crispr-Cas9 makes it easy, cheap, and fast to move genes around—any genes, in any living thing, from bacteria to people. “These are monumental moments in the history of biomedical research,” Baltimore says. “They don’t happen every day.”

Using the three-year-old technique, researchers have already reversed mutations that cause blindness, stopped cancer cells from multiplying, and made cells impervious to the virus that causes AIDS. Agronomists have rendered wheat invulnerable to killer fungi like powdery mildew, hinting at engineered staple crops that can feed a population of 9 billion on an ever-warmer planet. Bioengineers have used Crispr to alter the DNA of yeast so that it consumes plant matter and excretes ethanol, promising an end to reliance on petrochemicals. Startups devoted to Crispr have launched. International pharmaceutical and agricultural companies have spun up Crispr R&D. Two of the most powerful universities in the US are engaged in a vicious war over the basic patent. Depending on what kind of person you are, Crispr makes you see a gleaming world of the future, a Nobel medallion, or dollar signs.

The technique is revolutionary, and like all revolutions, it’s perilous. Crispr goes well beyond anything the Asilomar conference discussed. It could at last allow genetics researchers to conjure everything anyone has ever worried they would—designer babies, invasive mutants, species-specific bioweapons, and a dozen other apocalyptic sci-fi tropes. It brings with it all-new rules for the practice of research in the life sciences. But no one knows what the rules are—or who will be the first to break them.

IN A WAY, humans were genetic engineers long before anyone knew what a gene was. They could give living things new traits—sweeter kernels of corn, flatter bulldog faces—through selective breeding. But it took time, and it didn’t always pan out. By the 1930s refining nature got faster. Scientists bombarded seeds and insect eggs with x-rays, causing mutations to scatter through genomes like shrapnel. If one of hundreds of irradiated plants or insects grew up with the traits scientists desired, they bred it and tossed the rest. That’s where red grapefruits came from, and most barley for modern beer.

Genome modification has become less of a crapshoot. In 2002, molecular biologists learned to delete or replace specific genes using enzymes called zinc-finger nucleases; the next-generation technique used enzymes named TALENs.

Yet the procedures were expensive and complicated. They only worked on organisms whose molecular innards had been thoroughly dissected—like mice or fruit flies. Genome engineers went on the hunt for something better.

Scientists have used it to render wheat invulnerable to killer fungi. Such crops could feed billions of people.

As it happened, the people who found it weren’t genome engineers at all. They were basic researchers, trying to unravel the origin of life by sequencing the genomes of ancient bacteria and microbes called Archaea (as in archaic), descendants of the first life on Earth. Deep amid the bases, the As, Ts, Gs, and Cs that made up those DNA sequences, microbiologists noticed recurring segments that were the same back to front and front to back—palindromes. The researchers didn’t know what these segments did, but they knew they were weird. In a branding exercise only scientists could love, they named these clusters of repeating palindromes Crispr.

Then, in 2005, a microbiologist named Rodolphe Barrangou, working at a Danish food company called Danisco, spotted some of those same palindromic repeats in Streptococcus thermophilus, the bacteria that the company uses to make yogurt and cheese. Barrangou and his colleagues discovered that the unidentified stretches of DNA between Crispr’s palindromes matched sequences from viruses that had infected their S. thermophilus colonies. Like most living things, bacteria get attacked by viruses—in this case they’re called bacteriophages, or phages for short. Barrangou’s team went on to show that the segments served an important role in the bacteria’s defense against the phages, a sort of immunological memory. If a phage infected a microbe whose Crispr carried its fingerprint, the bacteria could recognize the phage and fight back. Barrangou and his colleagues realized they could save their company some money by selecting S. thermophilus species with Crispr sequences that resisted common dairy viruses.

As more researchers sequenced more bacteria, they found Crisprs again and again—half of all bacteria had them. Most Archaea did too. And even stranger, some of Crispr’s sequences didn’t encode the eventual manufacture of a protein, as is typical of a gene, but instead led to RNA—single-stranded genetic material. (DNA, of course, is double-stranded.)

That pointed to a new hypothesis. Most present-day animals and plants defend themselves against viruses with structures made out of RNA. So a few researchers started to wonder if Crispr was a primordial immune system. Among the people working on that idea was Jill Banfield, a geomicrobiologist at UC Berkeley, who had found Crispr sequences in microbes she collected from acidic, 110-degree water from the defunct Iron Mountain Mine in Shasta County, California. But to figure out if she was right, she needed help.

Luckily, one of the country’s best-known RNA experts, a biochemist named Jennifer Doudna, worked on the other side of campus in an office with a view of the Bay and San Francisco’s skyline. It certainly wasn’t what Doudna had imagined for herself as a girl growing up on the Big Island of Hawaii. She simply liked math and chemistry—an affinity that took her to Harvard and then to a postdoc at the University of Colorado. That’s where she made her initial important discoveries, revealing the three-dimensional structure of complex RNA molecules that could, like enzymes, catalyze chemical reactions.

The mine bacteria piqued Doudna’s curiosity, but when Doudna pried Crispr apart, she didn’t see anything to suggest the bacterial immune system was related to the one plants and animals use. Still, she thought the system might be adapted for diagnostic tests.

Banfield wasn’t the only person to ask Doudna for help with a Crispr project. In 2011, Doudna was at an American Society for Microbiology meeting in San Juan, Puerto Rico, when an intense, dark-haired French scientist asked her if she wouldn’t mind stepping outside the conference hall for a chat. This was Emmanuelle Charpentier, a microbiologist at Ume˚a University in Sweden.

As they wandered through the alleyways of old San Juan, Charpentier explained that one of Crispr’s associated proteins, named Csn1, appeared to be extraordinary. It seemed to search for specific DNA sequences in viruses and cut them apart like a microscopic multitool. Charpentier asked Doudna to help her figure out how it worked. “Somehow the way she said it, I literally—I can almost feel it now—I had this chill down my back,” Doudna says. “When she said ‘the mysterious Csn1’ I just had this feeling, there is going to be something good here.”

Back in Sweden, Charpentier kept a colony of Streptococcus pyogenesin a biohazard chamber. Few people want S. pyogenes anywhere near them. It can cause strep throat and necrotizing fasciitis—flesh-eating disease. But it was the bug Charpentier worked with, and it was in S. pyogenes that she had found that mysterious yet mighty protein, now renamed Cas9. Charpentier swabbed her colony, purified its DNA, and FedExed a sample to Doudna.

Working together, Charpentier’s and Doudna’s teams found that Crispr made two short strands of RNA and that Cas9 latched onto them. The sequence of the RNA strands corresponded to stretches of viral DNA and could home in on those segments like a genetic GPS. And when the Crispr-Cas9 complex arrives at its destination, Cas9 does something almost magical: It changes shape, grasping the DNA and slicing it with a precise molecular scalpel.

Here’s what’s important: Once they’d taken that mechanism apart, Doudna’s postdoc, Martin Jinek, combined the two strands of RNA into one fragment—“guide RNA”—that Jinek could program. He could make guide RNA with whatever genetic letters he wanted; not just from viruses but from, as far as they could tell, anything. In test tubes, the combination of Jinek’s guide RNA and the Cas9 protein proved to be a programmable machine for DNA cutting. Compared to TALENs and zinc-finger nucleases, this was like trading in rusty scissors for a computer-controlled laser cutter. “I remember running into a few of my colleagues at Berkeley and saying we have this fantastic result, and I think it’s going to be really exciting for genome engineering. But I don’t think they quite got it,” Doudna says. “They kind of humored me, saying, ‘Oh, yeah, that’s nice.’”

On June 28, 2012, Doudna’s team published its results in Science. In the paper and in an earlier corresponding patent application, they suggest their technology could be a tool for genome engineering. It was elegant and cheap. A grad student could do it.

The finding got noticed. In the 10 years preceding 2012, 200 papers mentioned Crispr. By 2014 that number had more than tripled. Doudna and Charpentier were each recently awarded the $3 million 2015 Breakthrough Prize. Time magazine listed the duo among the 100 most influential people in the world. Nobody was just humoring Doudna anymore.

MOST WEDNESDAY AFTERNOONS, Feng Zhang, a molecular biologist at the Broad Institute of MIT and Harvard, scans the contents of Scienceas soon as they are posted online. In 2012, he was working with Crispr-Cas9 too. So when he saw Doudna and Charpentier’s paper, did he think he’d been scooped? Not at all. “I didn’t feel anything,” Zhang says. “Our goal was to do genome editing, and this paper didn’t do it.” Doudna’s team had cut DNA floating in a test tube, but to Zhang, if you weren’t working with human cells, you were just screwing around.

That kind of seriousness is typical for Zhang. At 11, he moved from China to Des Moines, Iowa, with his parents, who are engineers—one computer, one electrical. When he was 16, he got an internship at the gene therapy research institute at Iowa Methodist hospital. By the time he graduated high school he’d won multiple science awards, including third place in the Intel Science Talent Search.

When Doudna talks about her career, she dwells on her mentors; Zhang lists his personal accomplishments, starting with those high school prizes. Doudna seems intuitive and has a hands-off management style. Zhang … pushes. We scheduled a video chat at 9:15 pm, and he warned me that we’d be talking data for a couple of hours. “Power-nap first,” he said.

If new genes that wipe out malaria also make mosquitoes go extinct, what will bats eat?

Zhang got his job at the Broad in 2011, when he was 29. Soon after starting there, he heard a speaker at a scientific advisory board meeting mention Crispr. “I was bored,” Zhang says, “so as the researcher spoke, I just Googled it.” Then he went to Miami for an epigenetics conference, but he hardly left his hotel room. Instead Zhang spent his time reading papers on Crispr and filling his notebook with sketches on ways to get Crispr and Cas9 into the human genome. “That was an extremely exciting weekend,” he says, smiling.

Just before Doudna’s team published its discovery in Science, Zhang applied for a federal grant to study Crispr-Cas9 as a tool for genome editing. Doudna’s publication shifted him into hyperspeed. He knew it would prompt others to test Crispr on genomes. And Zhang wanted to be first.

Even Doudna, for all of her equanimity, had rushed to report her finding, though she hadn’t shown the system working in human cells. “Frankly, when you have a result that is exciting,” she says, “one does not wait to publish it.”

In January 2013, Zhang’s team published a paper in Science showing how Crispr-Cas9 edits genes in human and mouse cells. In the same issue, Harvard geneticist George Church edited human cells with Crispr too. Doudna’s team reported success in human cells that month as well, though Zhang is quick to assert that his approach cuts and repairs DNA better.

That detail matters because Zhang had asked the Broad Institute and MIT, where he holds a joint appointment, to file for a patent on his behalf. Doudna had filed her patent application—which was public information—seven months earlier. But the attorney filing for Zhang checked a box on the application marked “accelerate” and paid a fee, usually somewhere between $2,000 and $4,000. A series of emails followed between agents at the US Patent and Trademark Office and the Broad’s patent attorneys, who argued that their claim was distinct.

A little more than a year after those human-cell papers came out, Doudna was on her way to work when she got an email telling her that Zhang, the Broad Institute, and MIT had indeed been awarded the patent on Crispr-Cas9 as a method to edit genomes. “I was quite surprised,” she says, “because we had filed our paperwork several months before he had.”

The Broad win started a firefight. The University of California amended Doudna’s original claim to overlap Zhang’s and sent the patent office a 114-page application for an interference proceeding—a hearing to determine who owns Crispr—this past April. In Europe, several parties are contesting Zhang’s patent on the grounds that it lacks novelty. Zhang points to his grant application as proof that he independently came across the idea. He says he could have done what Doudna’s team did in 2012, but he wanted to prove that Crispr worked within human cells. The USPTO may make its decision as soon as the end of the year.

The stakes here are high. Any company that wants to work with anything other than microbes will have to license Zhang’s patent; royalties could be worth billions of dollars, and the resulting products could be worth billions more. Just by way of example: In 1983 Columbia University scientists patented a method for introducing foreign DNA into cells, called cotransformation. By the time the patents expired in 2000, they had brought in $790 million in revenue.

It’s a testament to Crispr’s value that despite the uncertainty over ownership, companies based on the technique keep launching. In 2011 Doudna and a student founded a company, Caribou, based on earlier Crispr patents; the University of California offered Caribou an exclusive license on the patent Doudna expected to get. Caribou uses Crispr to create industrial and research materials, potentially enzymes in laundry detergent and laboratory reagents. To focus on disease—where the long-term financial gain of Crispr-Cas9 will undoubtedly lie—Caribou spun off another biotech company called Intellia Therapeutics and sublicensed the Crispr-Cas9 rights. Pharma giant Novartis has invested in both startups. In Switzerland, Charpentier cofounded Crispr Therapeutics. And in Cambridge, Massachusetts, Zhang, George Church, and several others founded Editas Medicine, based on licenses on the patent Zhang eventually received.

Thus far the four companies have raised at least $158 million in venture capital.

ANY GENE TYPICALLY has just a 50–50 chance of getting passed on. Either the offspring gets a copy from Mom or a copy from Dad. But in 1957 biologists found exceptions to that rule, genes that literally manipulated cell division and forced themselves into a larger number of offspring than chance alone would have allowed.

A decade ago, an evolutionary geneticist named Austin Burt proposed a sneaky way to use these “selfish genes.” He suggested tethering one to a separate gene—one that you wanted to propagate through an entire population. If it worked, you’d be able to drive the gene into every individual in a given area. Your gene of interest graduates from public transit to a limousine in a motorcade, speeding through a population in flagrant disregard of heredity’s traffic laws. Burt suggested using this “gene drive” to alter mosquitoes that spread malaria, which kills around a million people every year. It’s a good idea. In fact, other researchers are already using other methods to modify mosquitoes to resist the Plasmodium parasite that causes malaria and to be less fertile, reducing their numbers in the wild. But engineered mosquitoes are expensive. If researchers don’t keep topping up the mutants, the normals soon recapture control of the ecosystem.

Push those modifications through with a gene drive and the normal mosquitoes wouldn’t stand a chance. The problem is, inserting the gene drive into the mosquitoes was impossible. Until Crispr-Cas9 came along.

Today, behind a set of four locked and sealed doors in a lab at the Harvard School of Public Health, a special set of mosquito larvae of the African species Anopheles gambiae wriggle near the surface of shallow tubs of water. These aren’t normal Anopheles, though. The lab is working on using Crispr to insert malaria-resistant gene drives into their genomes. It hasn’t worked yet, but if it does … well, consider this from the mosquitoes’ point of view. This project isn’t about reengineering one of them. It’s about reengineering them all.

Kevin Esvelt, the evolutionary engineer who initiated the project, knows how serious this work is. The basic process could wipe out any species. Scientists will have to study the mosquitoes for years to make sure that the gene drives can’t be passed on to other species of mosquitoes. And they want to know what happens to bats and other insect-eating predators if the drives make mosquitoes extinct. “I am responsible for opening a can of worms when it comes to gene drives,” Esvelt says, “and that is why I try to ensure that scientists are taking precautions and showing themselves to be worthy of the public’s trust—maybe we’re not, but I want to do my damnedest to try.”

Esvelt talked all this over with his adviser—Church, who also worked with Zhang. Together they decided to publish their gene-drive idea before it was actually successful. They wanted to lay out their precautionary measures, way beyond five nested doors. Gene drive research, they wrote, should take place in locations where the species of study isn’t native, making it less likely that escapees would take root. And they also proposed a way to turn the gene drive off when an engineered individual mated with a wild counterpart—a genetic sunset clause. Esvelt filed for a patent on Crispr gene drives, partly, he says, to block companies that might not take the same precautions.

Within a year, and without seeing Esvelt’s papers, biologists at UC San Diego had used Crispr to insert gene drives into fruit flies—they called them “mutagenic chain reactions.” They had done their research in a chamber behind five doors, but the other precautions weren’t there.Church said the San Diego researchers had gone “a step too far”—big talk from a scientist who says he plans to use Crispr to bring back an extinct woolly mammoth by deriving genes from frozen corpses and injecting them into elephant embryos. (Church says tinkering with one woolly mammoth is way less scary than messing with whole populations of rapidly reproducing insects. “I’m afraid of everything,” he says. “I encourage people to be as creative in thinking about the unintended consequences of their work as the intended.”)

Ethan Bier, who worked on the San Diego fly study, agrees that gene drives come with risks. But he points out that Esvelt’s mosquitoes don’t have the genetic barrier Esvelt himself advocates. (To be fair, that would defeat the purpose of a gene drive.) And the ecological barrier, he says, is nonsense. “In Boston you have hot and humid summers, so sure, tropical mosquitoes may not be native, but they can certainly survive,” Bier says. “If a pregnant female got out, she and her progeny could reproduce in a puddle, fly to ships in the Boston Harbor, and get on a boat to Brazil.”

These problems don’t end with mosquitoes. One of Crispr’s strengths is that it works on every living thing. That kind of power makes Doudna feel like she opened Pandora’s box. Use Crispr to treat, say, Huntington’s disease—a debilitating neurological disorder—in the womb, when an embryo is just a ball of cells? Perhaps. But the same method could also possibly alter less medically relevant genes, like the ones that make skin wrinkle. “We haven’t had the time, as a community, to discuss the ethics and safety,” Doudna says, “and, frankly, whether there is any real clinical benefit of this versus other ways of dealing with genetic disease.”

Researchers in China announced they had used Crispr to edit human embryos.

That’s why she convened the meeting in Napa. All the same problems of recombinant DNA that the Asilomar attendees tried to grapple with are still there—more pressing now than ever. And if the scientists don’t figure out how to handle them, some other regulatory body might. Few researchers, Baltimore included, want to see Congress making laws about science. “Legislation is unforgiving,” he says. “Once you pass it, it is very hard to undo.”

In other words, if biologists don’t start thinking about ethics, the taxpayers who fund their research might do the thinking for them.

All of that only matters if every scientist is on board. A month after the Napa conference, researchers at Sun Yat-sen University in Guangzhou, China, announced they had used Crispr to edit human embryos. Specifically they were looking to correct mutations in the gene that causes beta thalassemia, a disorder that interferes with a person’s ability to make healthy red blood cells.

The work wasn’t successful—Crispr, it turns out, didn’t target genes as well in embryos as it does in isolated cells. The Chinese researchers tried to skirt the ethical implications of their work by using nonviable embryos, which is to say they could never have been brought to term. But the work attracted attention. A month later, the US National Academy of Sciences announced that it would create a set of recommendations for scientists, policymakers, and regulatory agencies on when, if ever, embryonic engineering might be permissible. Another National Academy report will focus on gene drives. Though those recommendations don’t carry the weight of law, federal funding in part determines what science gets done, and agencies that fund research around the world often abide by the academy’s guidelines.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

crispr new tools

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila(45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.

new-chin-year-dragon-02

Jakarta detik – Ilmuwan Amerika Serikat (AS) kini telah berhasil membikin genom (kumpulan gen) terkecil yang bisa hidup. Genom ini terdiri dari gen-gen dasar yang diperlukan bagi organisme untuk berfungsi dan bereproduksi sendiri.

Sebagaimana dilansir AFP, Sabtu (26/3/2016), ini adalah langkah besar menyibak misteri bagaimana kehidupan diciptakan. Genom sintetis dari bakteria ini punya julukan JCVI-syn3.0. Bakteri bikinan manusia ini hanya terdiri dari 473 gen, bandingkan dengan yang dipunya manusia, yakni terdiri dari 20.000 gen.

Pimpinan penelitian ini, Craig Venter, adalah orang pertama yang bisa menguraikan kode genom manusia. Selain Craig Venter, ada pula Clyde Hutchinson yang memimpin penelitian ini.

Meski begitu, tim ilmuwan ini belum menentukan fungsi dari 149 gen alias sepertiga dari total gen-gen yang mereka gabungkan itu. “Tugas pertama para investigator adalah menyelidiki aturan main gen-gen ini. Tugas ini menjanjikan pemahaman baru atas dasar kehidupan biologis,” kata Chris Voigt, ahli biologi sintetis dari Massachusetts Institute of Technology yang tak berpartisipasi dalam penelitian itu.

Sejumlah gen potensial yang berkesesuaian telah ditemukan di organisme lain. Ini mendorong mereka untuk mengkode protein universal dengan fungsi yang sampai sekarang masih belum bisa dipastikan.

Para peneliti itu menggunakan proses tes rancangan untuk mengidentifikasi gen quasi-esensial (gen yang sebenarnya tidak punya peran penting). Gen quasi-esensial itu dibutuhkan untuk pertumbuhan yang sehat, namun bukan untuk kehidupan.

Studi ini dipublikasikan pada jurnal ‘Science’ edisi Kamis (24/3). Melalui serangkaian eksperimen, mereka mencapai genom sintetis yang tereduksi, dibikin sekecil mungkin untuk menjamin tak ada lagi gen yang bisa mengacaukan genom ini.

Langkah Penting

“Satu-satunya  cara untuk menjawab pertanyaan mendasar tentang hidup adalah dengan cara mendapatkan genom paling minimal,” kata Craig Venter melalui telekonferensi.

“Mungkin satu-satunya jalan untuk melakukan itu adalah mencoba mensintesiskan (membikin -red) sebuah genom,” imbuh Venter.

24-Mar-2016                                        kehidupan sintetis

First Minimal Synthetic Bacterial Cell Designed and Constructed by Scientists at Venter Institute and Synthetic Genomics, Inc.

Cell, JCVI-syn3.0, was minimized to just 473 genes

(LA JOLLA, CA)—March 24, 2016—Researchers from the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) announced today the design and construction of the first minimal synthetic bacterial cell, JCVI-syn3.0.

Using the first synthetic cell, Mycoplasma mycoides JCVI-syn1.0 (created by this same team in 2010), JCVI-syn3.0 was developed through a design, build, and test process using genes from JCVI-syn1.0. The new minimal synthetic cell contains 531,560 base pairs and just 473 genes, making it the smallest genome of any organism that can be grown in laboratory media. Of these genes 149 are of unknown biological function. By comparison the first synthetic cell, M. mycoides JCVI-syn1.0 has 1.08 million base pairs and 901 genes.

A paper describing this research is being published in the March 25th print version of the journal Science by lead authors Clyde A. Hutchison, III, Ph.D. and Ray-Yuan Chuang, Ph.D., senior author J. Craig Venter, Ph.D., and senior team of Hamilton O. Smith, MD, Daniel G. Gibson, Ph.D., and John I. Glass, Ph.D.

“Our attempt to design and create a new species, while ultimately successful, revealed that 32% of the genes essential for life in this cell are of unknown function, and showed that many are highly conserved in numerous species. All the bioinformatics studies over the past 20 years have underestimated the number of essential genes by focusing only on the known world. This is an important observation that we are carrying forward into the study of the human genome,” said Dr. Venter, Founder, Executive Chairman, and CEO, JCVI.

The research to construct the first minimal synthetic cell at JCVI was the culmination of 20 years of research that began in 1995 after the genome sequencing of the first free-living organism, Haemophilus influenza, followed by the sequencing of Mycoplasma genitalium. A comparison of these two genomes revealed a common set of 256 genes which the team thought could be a minimal set of genes needed for viability. In 1999 Dr. Hutchison led a team who published a paper describing the use of global transposon mutagenesis techniques to identify the nonessential genes in M. genitalium.

Over the last 50 years more than 2,000 publications have contemplated minimal cells and their use in elucidating first principals of biology. From the start, the goal of the JCVI team was similar—build a minimal operating system of a cell to understand biology but to also have a desirable chassis for use in industrial applications. The creation of the first synthetic cell in 2010 did not inform new genome design principles since the M. mycoides genome was mostly recapitulated as in nature.  Rather, it established a work flow for building and testing whole genome designs, including a minimal cell, from the bottom up starting from a genome sequence.

To create JCVI-syn3.0, the team used an approach of whole genome design and chemical synthesis followed by genome transplantation to test if the cell was viable. Their first attempt to minimize the genome began with a simple approach using information in the biochemical literature and some limited transposon mutagenesis work, but this did not result in a viable genome. After improving transposon methods, they discovered a set of quasi-essential genes that are necessary for robust growth which explained the failure of their first attempt.

To facilitate debugging of non-functional reduced genome segments, the team built the genome in eight segments at a time so that each could be tested separately before combining them to generate a minimal genome. The team also explored gene order and how that affects cell growth and viability, noting that gene content was more critical to cell viability than gene order. They went through three cycles of designing, building, and testing ensuring that the quasi-essential genes remained, which in the end resulted in a viable, self-replicating minimal synthetic cell that contained just 473 genes, 35 of which are RNA-coding. In addition, the cell contains a unique 16S gene sequence.

The team was able to assign biological function to the majority of the genes with 41% of them responsible for genome expression information, 18% related to cell membrane structure and function, 17% related to cytosolic metabolism, and 7% preservation of genome information. However, a surprising 149 genes could not be assigned a specific biological function despite intensive study. This remains an area of continued work for the researchers.

“This paper represents more than five years of work by an amazingly talented group of people. Our goal is to have a cell for which the precise biological function of every gene is known,” said Dr. Hutchison, Distinguished Professor, JCVI.

The team concludes that a major outcome of this minimal cell program are new tools and semi-automated processes for whole genome synthesis.  Many of these synthetic biology tools and services are commercially available through SGI and SGI-DNA including a synthetic DNA construction service specializing in building large and complex DNA fragments including combinatorial gene libraries, Archetype® genomics software, Gibson Assembly® kits, and the BioXp™, which is a benchtop instrument for producing accurate synthetic DNA fragments.

“This paper signifies a major step toward our ability to design and build synthetic organisms from the bottom up with predictable outcomes.  The tools and knowledge gained from this work will be essential to producing next generation production platforms for a wide range of disciplines,” said Dr. Gibson, Vice President, DNA Technologies, SGI; Associate Professor, JCVI.

The other researchers on this paper have been integral to this work for much of the last decade. Current and former JCVI and SGI scientists are: Chuck Merryman, Ph.D., Ray-Yuan Chuang, Ph.D., Vladimir Noskov, Ph.D., Nacyra Assad-Garcia, John Gill, Krishna Kannan, Ph.D., Bogumil Karas, Ph.D., Li Ma, Zhi-Qing Qi, Ph.D., R. Alexander Richter, Ph.D., Lijie Sun, Ph.D., Yo Suzuki, Ph.D., Billyana Tsvetanova, Ph.D. and Kim Wise, Ph.D.

Other authors on the paper are: Thomas J. Deerinck and Mark H. Ellisman, Ph.D., University of California, San Diego National Center for Microscopy and Imaging Research; James F. Pelletier, Center for Bits and Atoms and Department of Physics, Massachusetts Institute of Technology; Elizabeth A. Strychalski, National Institute of Standards and Technology.

This work was funded by SGI, the JCVI endowment and the Defense Advanced Research Projects Agency’s Living Foundries program, HR0011-12-C-0063.

About J. Craig Venter Institute

The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 200 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The JCVI is a 501 (c)(3) organization. For additional information, please visit http://www.JCVI.org.

JCVI Media Contact

Heather Kowalski, hkowalski(AT)jcvi.org

– See more at: http://www.jcvi.org/cms/press/press-releases/full-text/article/first-minimal-synthetic-bacterial-cell-designed-and-constructed-by-scientists-at-venter-institute-an/#sthash.kTm3YrGS.dpuf

rose KECIL

DNA Robots Programmed to Kill Cancer Cells, Harvard Study Shows

Usaha ini mengantarkan ilmuwan kepada Mycoplasma, yakni bakteri dengan sel genom terkecil. Mycoplasma dapat mereplikasi (menggandakan) dirinya sendiri secara otonom.

“Bila Anda tak tahu apapun soal pesawat terbang dan Anda sedang melihat Boeing 777, dan Anda cuma mencari tahu fungsi bagian-bagian pesawat itu dengan cara mengurainya, dan anda mempreteli mesinnya dari sayap kanan, pesawat itu masih bisa terbang dan mendarat,” kata Venter mencoba menjelaskan dengan analogi.

“Jadi mungkin Anda mengatakan ini adalah komponen yang tidak penting, dan Anda tak benar-benar menemukan hal yang paling esensial sampai anda mempreteli (mesin pesawat) yang satunya lagi. Dan itulah yang terjadi, lagi dan lagi, pada biologi saat kami akan mendapati komponen yang non-esensial, sampai pada saat kita mempreteli pasangannya,” kata Venter.

Pada satu penemuan penting, tim peneliti belajar bahwa beberapa gen digolongkan sebagai ‘non-esensial (tidak penting)’. Artinya, gen satunya lagi adalah gen yang wajib diperlukan, alias gen yang ‘esensial’, atau gen minimal yang dibutuhkan.

Genom minimal tak punya gen yang bisa memodifikasi dan membatasi DNA. Genom minimal juga kurang akan gen yang mengkode lipoprotein.

Bagaimanapun juga, ‘genom minimalis’ ini mengandung semua gen yang diperlukan dalam pembacaan dan mengekspresikan informasi genetik dalam genom, sebagaimana dalam hal menjaga informasi genetik dari generasi ke generasi.

(dnu/dnu)

animated-rocket-and-space-shuttle-image-0026

By Elizabeth Lopatto – Feb 16, 2012

Scientists have created a robot made entirely from DNA that can be instructed to find diseased cells in the body and deliver a payload to kill or reprogram them, according to a study from Harvard University.

The robot was constructed by folding DNA strands into a shape that looks roughly like a clamshell. The researchers programmed the nano-sized device to open in the presence of leukemia and lymphoma cells in a laboratory dish, where they delivered immune system antibodies that caused the cells to self-destruct, according to a report in the journal Science.

The next step will be to test the system in animals, tweaking the robot so that it can circulate longer in the blood to locate all cancer cells. The technology isn’t yet ready for commercial use, said Shawn Douglas, an author of the study.

“In diseases such as cancer we know if we can find every single last cell and kill or reprogram it, we can cure that disease,” said Douglas, a researcher at the Wyss Institute for Biologically Inspired Engineering at Harvard, in Boston. “A lot of our current therapies fall short.”

The idea is based on the behavior of the body’s immune cells, which recognize viruses or other invaders and attack them, Douglas said. The DNA nano-robots, with similar capabilities, may potentially lead to the development of new types of targeted cancer treatments that kill only abnormal cells, he said.

The robots don’t reproduce. They have to be constructed in a process that has gained traction since the idea of DNA nanotechnology was first suggested in 1982.
Genetic Information

DNA is a material, shaped in the form of a revolving ladder, which carries the genetic information in our cells.

The double-sided strands have so-called sticky ends that allow them to be joined together with other DNA. Scientists, led by Nadrian Seeman, now head of the Department of Chemistry at New York University, have used those sticky ends to form DNA into lattices that can be shaped in various ways.

The latest research created a robot in a clamshell shape that’s held together with a “zipper” constructed of a special sequence of DNA, the report said. The zipper was programmed to release its grip when it recognized specific targets on a cell, allowing the robot to release its payload.

In the experiment, Douglas and his fellow scientists used the robot they constructed to deliver instructions encoded in antibodies to the cancer cells.

“It’s an important step forward in specific targeting,” said Milan Stojanovic, an assistant professor of experimental therapeutics at Columbia University in New York who wasn’t involved in the research, in an e-mail. “It looks very exciting.”

Besides cancer, the robots may also benefit people with autoimmune disease, Douglas said. One day, the robots might be used to find immune cells wrongly attacking the body and reprogram them, he said.

Juni 23, 2016

playboy: PENGOBATAN kanker PAYUDARA ala amrik … 020911_240616

Filed under: Medicine — bumi2009fans @ 2:01 pm

Penatalaksanaan Kanker Payudara dengan Denosumab

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Liputan6.com, Jakarta Sebuah studi baru menunjukkan wanita yang memiliki gen BRCA1 yang rusak berisiko meningkatkan kanker payudara, dan ovarium hingga 87 persen. Seperti yang dialami Angelina Jolie yang sudah melakukan mastektomi ganda pada 2013 lalu. Setelah itu, ovariumnya pun diangkat karena kanker sudah menyerang.

Prosedur drastis seperti itu mungkin tidak diperlukan lagi. Karena sudah ada obat untuk mencegah tumor yang dapat membentuk jaringan di payudara, peneliti mengklaim.

Peneliti Australia yang melakukan tes laboratorium mengatakan obat osteoporosisdenosumab, yang biasa digunakan untuk mengobati penyakit tulang, juga efektif melawan kanker payudara. Sehingga wanita tidak harus melalui operasi yang menyakitkan untuk mencegah kanker.

Obat tersebut sudah diuji pada manusia, sehingga waktunya tak lama sebelum obat itu diproduksi lagi. Dibandingkan dengan obat lain yang masih harus menjalani uji keamanan klinis yang panjang, dikutip lamanDailymail, Selasa (21/6/2016).

Para peneliti di Institut Walter dan Eliza Hall di Melbourne mengatakan denosumab dicoba dengan menggunakan sel pra-kanker di jaringan payudara. Hasilnya, tumor yang terbentuk tak berkembang. Mereka mengatakan bahwa dalam studi klinis, obat bisa memberikan alternatif non-bedah untuk menghentikan kanker payudara pada yang berisiko tinggi.

Peneliti Linda Nolan, The University of Melbourne mengatakan mereka telah mengidentifikasi sel-sel payudara yang berpotensi tinggi menjadi kanker jika obat dihentikan.

“Sel-sel ini berkembang biak dengan cepat, dan rentan terhadap rusaknya DNA. Kami antusias sekali dengan penemuan bahwa sel-sel pra-kanker dapat diidentifikasi oleh protein penanda yang disebut RANK,” ujarnya.

Profesor Lindeman, yang juga seorang ahli onkologi medis di Rumah Sakit The Royal Melbourne menambahkan, “Kami pikir strategi ini dapat menunda atau mencegah kanker payudara pada wanita dengan mutasi gen BRCA1 yang diwariskan. Sebuah uji klinis mulai dilakukan lebih lanjut. Penemuan ini sangat penting bagi wanita yang memiliki gen BRCA1 yang rusak.”

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latimes.com

Breast cancer: Six women, six paths

By Amanda Mascarelli, Special to the Los Angeles Times

October 1, 2011

advertisement

Breast cancer is no longer considered a single disease. New molecular tools are allowing doctors to see what is going on inside tumors with much greater accuracy, enabling them to tailor their therapeutic approach to fit the traits of each cancer and the needs of each patient, as the women below illustrate.

Sailing through

Name: Caryl Engstrom

Current age: 51

Home: Los Angeles

Diagnosis: Stage 2B breast cancer that was ER-positive

Age at diagnosis: 49

Engstrom had a mastectomy, followed by five months of a combination chemotherapy known as ACT (which includes the drugs Adriamycin, Cytoxan and Taxotere) and 61/2 weeks of radiation. She now takes Tamoxifen, an estrogen-blocking pill, to reduce her risk of recurrence. She is cancer-free.

“My feeling is that the minute I had my surgery, I was cancer-free. The chemo and the radiation are what I call the insurance policy. For me it was about making decisions based on what’s going to give me the best possible chance of having a cancer-free life.

“I know it sounds crazy, but I never felt like, ‘This is gonna kill me.’ It was like, ‘Let’s figure out how to make this work.’ My oncologist told me to have my normal life — every three weeks you’re going to have this treatment, you’re not going to feel well for a few days but you’ll bounce back. It was completely manageable. If you have a healthy diet and a healthy mindset, you exercise, you have a good support system, good friends, you sail through it.”

Gentler choices

Name: Christina Eason

Current age: 37

Home: Thousand Oaks, Calif.

Diagnosis: Stage 1 ductal carcinoma in situ (DCIS) that was ER-positive and PR-positive

Age at diagnosis: 32

Eason’s type of tumor carries an excellent prognosis —96% to 98% of women are alive 10 years after their diagnosis, according to the National Institutes of Health. Eason had a lumpectomy to remove her tumor but, against the advice of her doctor, she did not have a radical mastectomy, radiation or follow-up drug treatments. (Chemotherapy wasn’t recommended because her tumor was localized). Instead, Eason relies on a diet of what she calls “nutrition therapy,” which includes alternating between a vegetarian and vegan diet and doing an annual “detoxification” that involves drinking nothing but specialized juices for three to 10 days. She also takes a combination of vitamin, mineral and herbal supplements to maintain her cancer-free status.

“I didn’t just do chemo and walk away. I have a battle ahead of me for the rest of my life to make choices about what I eat and how I am affecting the estrogen in my body. It’s a daily thing. I want to be around for the next 50 years for my kids, so what am I going to do to make sure that happens?

“I’ve just come out of a season of dealing with paralyzing fear — of learning to cope by controlling where my mind is going. It’s a lot of prayer. My life isn’t guaranteed from this day to the next.”

Tackling high risk

Name: Clare Hobby

Current age: 41

Home: Houston

Diagnosis: Stage 2 ductal carcinoma that was ER-positive and HER2 positive

Age at diagnosis: 38

Hobby was diagnosed in 2008 and then discovered she had a mutation in her BRCA1 gene that increased her risk of developing breast cancer by 55% to 87%. Now that she has had one tumor, she is at greater risk of developing a second primary breast cancer. Hobby had chemotherapy, radiation and a double mastectomy, among other surgeries. As a preventive measure, she had her ovaries and uterus removed last year. She is also taking a drug called Arimidex to kill any stray cells that may have evaded her chemo and radiation treatments and to block her body’s production of estrogen, a hormone that can feed breast cancer tumors.

“I feel fortunate that due to this individualized approach I know more about my cancer and can take more active steps in working preventively with my children, who may have also inherited the gene. Individualized medicine has been a huge gift for us because we can talk to our kids in a completely different way.”

The lonely fight

Name: Faina Sechzer

Current age: 59

Home: Princeton, N.J.

Diagnosis: Stage 2 breast cancer that was ER-positive

Age at diagnosis: 57

Sechzer’s cancer was discovered at an early stage, but a lumpectomy revealed that it had spread to a lymph node, requiring a second surgery. She opted for chemotherapy with the drugs Cytoxan and Taxotere and also received three weeks of radiation treatment. She is cancer-free and is in the midst of a five-year course of Arimidex to suppress her body’s production of estrogen.

“After all this research, I ended up making decisions out of my gut and out of knowing myself. I wanted to live my life knowing that whatever there was to do, I did.

“The biggest difficulty was not physical pain but emotional pain. You find yourself very lonely at that time. I have a loving husband, loving children and a loving support group. But you still find yourself very lonely.”

Hitting on the right drug

Name: Margaret Mauran Zuccotti

Current age: 42

Home: Jenkintown, Pa.

Diagnosis: Stage 4 inflammatory breast cancer that was HER2-positive

Age at diagnosis: 37

Zuccotti’s cancer had spread to her liver and a bone near her right eye socket. She started a tailored treatment plan that included eight months of chemotherapy using a combination of Taxol and Herceptin. She also had a mastectomy. Zuccotti has been cancer-free since July 2007. She continues to be treated with Herceptin every three weeks to keep her cancer from returning.

“I feel very hopeful because my response has been so positive. For someone with my diagnosis and my markers, Herceptin is completely life-changing. I have a cousin who was in her early 30s when she got a similar diagnosis. She was pre-Herceptin. They tried many different medicines. They’re all really hard on the system, on the heart and the spirit. To have something that fixes this particular cancer and seems to be doing it incredibly well is wonderful. It gives you such hope when you’re in this sunny subgroup, where before there was no alternative but to cross your fingers and hope your life expectancy could be extended.”

Blossoming again

Name: Tina Roark

Current age: 49

Home: Eldorado, Ark.

Diagnosis: Stage 3 ductal cell carcinoma that was ER-positive

Age at diagnosis: 37

After getting her diagnosis, Roark found out she had a BRCA1 mutation. She had a bilateral mastectomy, three chemotherapy treatments and took the anti-estrogen drug Tamoxifen for five years to reduce the risk of recurrence. The treatments kept her cancer at bay for several years, but then it spread to her bones, spine, liver, lungs, ribs, hip and sternum. She tried three more chemotherapy treatments that failed. Now she is participating in a clinical trial of a drug called olaparib that appears to have anti-tumor effects in people with BRCA1 and BRCA2 mutations who have advanced-stage breast or ovarian cancer. Her tumors have shrunk dramatically since she began taking olaparib in March.

“I was done. My oncologist had just seen me wither away. It was just time to give up. I had felt so bad. I didn’t have any quality of life. You’re surviving, but is it worth surviving?

“It’s like I’ve blossomed again, from this wilted flower to this big standing up, face-the-world situation now. If it works six months or a year or 10 years — however long it works — it’s a bonus for me.”

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Juni 5, 2016

jatuhAT1: protein yang mencur1 panggung “the GREATEST”

Filed under: GLOBAL ECONOMY,Medicine — bumi2009fans @ 12:09 am

ninds: What causes the disease?

Parkinson’s disease occurs when nerve cells, or neurons, in the brain die or become impaired.  Although many brain areas are affected, the most common symptoms result from the loss of neurons in an area near the base of the brain called thesubstantia nigra.  Normally, the neurons in this area produce an important brain chemical known as dopamine.  Dopamine is a chemical messenger responsible for transmitting signals between the substantia nigra and the next “relay station” of the brain, the corpus striatum, to produce smooth, purposeful movement.  Loss of dopamine results in abnormal nerve firing patterns within the brain that cause impaired movement.  Studies have shown that most people with Parkinson’s have lost 60 to 80 percent or more of the dopamine-producing cells in the substantia nigra by the time symptoms appear, and that people with PD also have loss of the nerve endings that produce the neurotransmitter norepinephrine. Norepinephrine, which is closely related to dopamine, is the main chemical messenger of the sympathetic nervous system, the part of the nervous system that controls many automatic functions of the body, such as pulse and blood pressure.  The loss of norepinephrine might explain several of the non-motor features seen in PD, including fatigue and abnormalities of blood pressure regulation.

The affected brain cells of people with PD contain Lewy bodies—deposits of the protein alpha-synuclein.  Researchers do not yet know why Lewy bodies form or what role they play in the disease.  Some research suggests that the cell’s protein disposal system may fail in people with PD, causing proteins to build up to harmful levels and trigger cell death.  Additional studies have found evidence that clumps of protein that develop inside brain cells of people with PD may contribute to the death of neurons.  Some researchers speculate that the protein buildup in Lewy bodies is part of an unsuccessful attempt to protect the cell from the toxicity of smaller aggregates, or collections, of synuclein.

Genetics.  Scientists have identified several genetic mutations associated with PD, including the alpha-synuclein gene, and many more genes have been tentatively linked to the disorder.  Studying the genes responsible for inherited cases of PD can help researchers understand both inherited and sporadic cases.  The same genes and proteins that are altered in inherited cases may also be altered in sporadic cases by environmental toxins or other factors.  Researchers also hope that discovering genes will help identify new ways of treating PD.

Environment.  Exposure to certain toxins has caused parkinsonian symptoms in rare circumstances (such as exposure to MPTP, an illicit drug, or in miners exposed to the metal manganese).  Other still-unidentified environmental factors may also cause PD in genetically susceptible individuals.

Mitochondria.  Several lines of research suggest that mitochondria may play a role in the development of PD.  Mitochondria are the energy-producing components of the cell and abnormalities in the mitochondria are major sources of free radicals—molecules that damage membranes, proteins, DNA, and other parts of the cell. This damage is often referred to as oxidative stress.  Oxidative stress-related changes, including free radical damage to DNA, proteins, and fats, have been detected in the brains of individuals with PD.  Some mutations that affect mitochondrial function have been identified as causes of PD.

While mitochondrial dysfunction, oxidative stress, inflammation, toxins, and many other cellular processes may contribute to PD, the actual cause of the cell loss death in PD is still undetermined.

What genes are linked to Parkinson’s disease?

Several genes have been definitively linked to PD.  The first to be identified was alpha-synuclein.  In the 1990s, researchers at National Institutes of Health and other institutions studied the genetic profiles of a large Italian family and three Greek families with familial PD and found that their disease was related to a mutation in this gene.  They found a second alpha-synuclein mutation in a German family with PD.  These findings prompted studies of the role of alpha-synuclein in PD, which led to the discovery that Lewy bodies seen in all cases of PD contain alpha-synuclein protein.  This discovery revealed the link between hereditary and sporadic forms of the disease.

In 2003, researchers studying inherited PD discovered that the disease in one large family was caused by a triplication of the normal alpha-synuclein gene on one copy of chromosome 4 (a chromosome is a threadlike structure of a protein and the genetic material DNA).  This triplication caused people in the affected family to produce too much of the normal alpha-synuclein.  This study showed that an excess of the normal form of synuclein could result in PD, just as the abnormal form does.

Other genes linked to PD include parkin, DJ-1, PINK1, and LRRK2. DJ-1 and PINK1 cause rare, early-onset forms of PD.  The parkin gene is translated into a protein that normally helps cells break down and recycle proteins.  DJ-1 normally helps regulate gene activity and protect cells from oxidative stress.  PINK1 codes for a protein active in mitochondria.  Mutations in this gene appear to increase susceptibility to cellular stress.

Mutations in LRRK2 were originally identified in several English and Basque families as a cause of a late-onset PD.  Subsequent studies have identified mutations of this gene in other families with PD as well as in a small percentage of people with apparently sporadic PD.  LRRK2 mutations are a major cause of PD in North Africa and the Middle East.

Another interesting association is with the GBA gene, which makes the enzyme glucocerebrosidase.  Mutations in both GBA genes cause Gaucher disease (in which fatty acids, oils, waxes, and steroids accumulate in the brain), but different changes in this gene are associated with an increased risk for Parkinson’s disease as well.  Investigators seek to understand what this association can tell us about PD risk factors and potential treatments.

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nih senior health: Structure of Alpha-Synuclein Protein Takes Surprising Twist

Researchers recently discovered that alpha-synuclein (α-synuclein) — a proteinthat forms clumps called Lewy bodies in the brain cells of people with Parkinson’s disease (PD)— exists in cells in a radically different form than they previously thought. This finding provides new clues about the causes of Parkinson’s, as well as suggests new strategies for treatment. The study was published in the August 14, 2011 issue of Nature.

By studying the human brain, scientists have been able to link Lewy bodies with the brain cell death that triggers PD. Although scientists do not know exactly what causes α-synuclein to form toxic protein clumps, the answer almost certainly has to do with changes in the protein’s structure.

Most proteins, which are composed of long chains of amino acids, fold upon themselves into complex three-dimensional structures that give them their unique properties. Until now, researchers thought that α-synuclein was one of the minority of proteins in cells that existed as a floppy, unfolded chain. This might have helped to explain the protein’s tendency to form clumps.

But researchers led by Dennis Selkoe, M.D., at Harvard Medical School questioned this school of thought, wondering if α-synuclein only appeared to be unstructured because of the methods scientists were using to study it. Most previous studies used α-synuclein produced in bacteria or analyzed under harsh conditions that could disrupt the natural protein structure. So Dr. Selkoe and his colleagues studied α-synuclein purified from human cells using several gentler methods.

Results

  • Instead of being unfolded, α-synuclein in human cells adopts a twisted structure known as an α-helix.
  • Four of these α-helical protein molecules join together to form a tetramer in cells.
  • The α-synuclein tetramer did not clump together like the single, unfolded α-synuclein molecule that scientists had studied previously.

What Does it Mean?

A protein’s structure provides valuable clues to its function. Scientists still do not know exactly what role α-synuclein plays in healthy cells. However, α-synuclein deposits are the hallmark of the pathological changes in brains of people with PD. Many scientists believe that if we find a way to prevent its accumulation, we may be able to stop and potentially reverse the damage these deposits may cause to the brain. Therefore, a better understanding of α-synuclein’s structure and function might help scientists figure out what causes the protein to form Lewy bodies in PD.

Importantly, the α-synuclein tetramer resisted clumping together. This result suggests that in PD, the tetramer may fall apart into individual α-synuclein molecules that can then cluster into Lewy bodies. By designing new drugs that stabilize the α-synuclein tetramer, scientists might someday be able to reduce the formation of Lewy bodies in people with PD.

Reference: Bartels, T., Choi, J. G., & Selkoe, D. J. (2011). α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation Nature, 477(7362), 107–110. doi:10.1038/nature10324  PubMed PMID: 21841800; PubMedCentral PMCID: PMC3166366.

Source Date: Sep 19 2011

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biomed central: α-Synuclein is a small protein that has special relevance for understanding Parkinson disease and related disorders. Not only is α-synuclein found in Lewy bodies characteristic of Parkinson disease, but also mutations in the gene for α-synuclein can cause an inherited form of Parkinson disease and expression of normal α-synuclein can increase the risk of developing Parkinson disease in sporadic, or non-familial, cases. Both sporadic and familial Parkinson disease are characterized by substantial loss of several groups of neurons, including the dopaminergic cells of the substantia nigra that are the target of most current symptomatic therapies. Therefore, it is predicted that α-synuclein, especially in its mutant forms or under conditions where its expression levels are increased, is a toxic protein in the sense that it is associated with an increased rate of neuronal cell death. This review will discuss the experimental contexts in which α-synuclein has been demonstrated to be toxic. I will also outline what is known about the mechanisms by which α-synuclein triggers neuronal damage, and identify some of the current gaps in our knowledge about this subject. Finally, the therapeutic implications of toxicity of α-synuclein will be discussed.

All neurodegenerative diseases share the common phenomenon that neurons, usually relatively specific groups, are lost progressively as the disease develops. In some cases, we can provide partial relief for patients by treating some of their symptoms. However, because we don’t understand the underling mechanisms of why neurons die, degeneration continues inexorably and old symptoms often become unresponsive while new ones arrive. At the end of the disease process, we are left with only a few clues about what might have happened based on what we can glean from the pathology of the disease using post mortem samples. In general, the root cause of neurodegeneration remains obscure although rare genetic variants are helpful in that we can be certain that an inherited mutation acts as the trigger for disease in that specific family.

Here, I will discuss cell loss related to Parkinson disease (PD) in the context of one protein, α-synuclein, that has several links to the disorder. In doing so, I will outline what we know about the ways in which a protein can lead to cell death. Before doing so, it is worth discussing what PD is, and what it isn’t.

Cell death in PD

It is very commonly said that PD is the second most common neurodegenerative disease and results from a loss of dopamine neurons. The first fact is dull and the second tells only part of the story. It is true that PD patients have a substantial loss of dopamine in the striatum resulting from a relatively selective loss of dopaminergic projection neurons in the substantia nigra pars compacta. Both biochemical measures and imaging modalities suggest that at least a 70% decrease in striatal dopamine occurs before the onset of clinical parkinsonism and progresses over time [1]. These estimates of the extent of striatal dopamine depletion, combined with the observation that the majority of dopaminergic neurons are lost by the end of the disease process, imply that there is substantial cell death throughout the PD disease process. It is not possible to show this directly, but measurements of nigral cell numbers in neurologically normal people and in non-human primates reveal a slow progressive loss of dopamine neurons with age [2]. In this view, parkinsonism is an accelerated, but still slow, cell death phenotype that would normally be seen with aging [3].

However, while there is relative vulnerability of dopaminergic neurons in the substantia nigra [4], not all dopamine cells are affected in PD. For example, although dopaminergic neurons in the ventral tegmental area that project to the nucleus accumbens do degenerate [5], compared to the dopaminergic neurons in the substantia nigra pars compacta these cells are relatively spared [6, 7].

Furthermore, not all affected neurons in PD are dopaminergic. Non-motor symptoms are a serious problem for many PD patients and are often untreated by replacement therapy with L-DOPA (3,4-dihydroxy-L-phenylalanine) [8]. A good example of non-dopaminergic cells that degenerate in PD is the cholinergic neurons in the dorsal vagal nucleus [9]. It has been suggested that involvement of non-nigral regions underlies the complex clinical picture in PD [10]. Therefore, although there is some specificity to cell death in PD, there is no absolute selectivity for any specific neurotransmitter group or anatomic region. It is also important to note that loss of nigral neurons occurs in diverse pathological situations [4] and that on its own, nigral cell loss defines the clinical term parkinsonism, not Parkinson disease.

This distinction is also important when discussing the other major pathological event in PD that appears alongside cell death, the formation of Lewy bodies and Lewy neurites. Lewy bodies are intracellular deposits of proteins and lipids [11] that were traditionally stained with eosin but now are more sensitively recognized by antibodies to specific marker proteins [12]. Using electron microscopy, Lewy bodies are fibrillar structures with a recognizable core and halo [13]. The range of Lewy pathology is now recognized as encompassing many regions of the diseased brain [14] including, for example, the olfactory bulb, raphe nucleus, locus coeruleus and the basal nucleus of Meynert. Additionally, some reports suggest that the nigra is not the first place where Lewy bodies form [15]. How this relates to the extent of cell loss in each region is not well defined. Lewy bodies are also seen in dementia with Lewy bodies (DLB, also known as Diffuse Lewy body Disease or DLBD), suggesting that PD and DLBD are related to one another by shared pathology and maybe by shared etiology.

Therefore, PD is a disease where substantial cell loss in the nigra occurs alongside the formation of Lewy bodies. Neither cell loss nor Lewy bodies is absolutely specific for PD but both are required for a diagnosis of PD under current definitions [16]. This review will focus on cell death, but it is important to understand a little more about the most commonly used marker for Lewy bodies; α-synuclein.

α-Synuclein is a marker of the PD process

The first member of the family of proteins for which α-synuclein is named was cloned from the neuromuscular junction of the electric eel [17]. Antibodies against that protein labeled both synapses and nuclei, leading to the naming of synuclein. A related protein was cloned from zebra finch as a protein upregulated during the process of song learning, a period of enormous synaptic plasticity [18]. In humans, there are three synuclein family members (α-,β-,γ-) and all synuclein genes are relatively well conserved both within and between species [19]. The synuclein genes are specific to the vertebrate lineage in that neither single cell organisms (including yeast) nor invertebrates (Drosophila melanogaster, Caenorhabditis elegans) have any apparent synuclein homologue. Additionally, primate α-synuclein sequences differ from other vertebrate synucleins by a substitution of Alanine for a Threonine at position 53 [20]. These two interesting facts about the evolutionary relationships in the synuclein family are important for understanding some of the experimental systems where synuclein has been explored.

The normal function of α-synuclein is poorly understood. Although it is expressed at high levels in the brain, relatively specifically within neurons, it is also found in other tissues, e.g., hematopoietic cells [21, 22]. α-Synuclein can bind to lipids [23] and, in neurons, is associated with presynaptic vesicles [24, 25] and the plasma membrane, possibly via lipid rafts [26]. The association of α-synuclein with vesicles is modulated by synaptic activity where the protein dissociates from vesicles after electrical stimulation of the neuron and only slowly re-associates [27]. However, α-synuclein knockout mice show only subtle abnormalities in neurotransmission [28, 29, 30], suggesting that α-synuclein plays a non-essential function at the synapse. There is some evidence that such a modulatory role may be more important under conditions where reactive oxygen species or nitric oxide are present [31, 32], although the mechanism(s) are not yet fully defined.

In the normal brain, α-synuclein immunostaining reveals a diffuse pattern of reactivity throughout the neuropil that would be consistent with a predominantly synaptic localization [25]. However, in PD brains, α-synuclein antibodies strongly stain Lewy bodies [33] and Lewy neurites [34]. Because of this sensitivity, α-synuclein staining is now more commonly used than eosin or ubiquitin staining for these structures. Biochemical analyses have shown that α-synuclein is a major protein component of Lewy bodies and may be part of the fibrillar structure of these structures [35]. The deposited, pathological forms of α-synuclein are aggregated and show lower solubility than the normal protein [36] and may be modified post-translationally by truncation, nitration, ubiquitylation and phosphorylation [37, 38, 39, 40].

Therefore, α-synuclein protein deposition into Lewy bodies is a marker of the PD disease state. However, because we require Lewy bodies for a PD diagnosis this isn’t an especially strong argument for involvement of α-synuclein in the disease process. It is also important to note that, although we cannot determine if Lewy bodies previously formed in the cells that eventually died, the individual neurons where Lewy bodies are found are the ones that have survived the disease process (though they still may be dysfunctional). Very recently, it has been shown that Lewy bodies form in functional dopaminergic neurons grafted in to brains of people with PD as a therapeutic intervention [41, 42], although this is not always seen [43]. These were embryonic cells that remained apparently healthy and were functional after grafting, which suggests that there is the environment of the PD brain predisposes even young cells to form Lewy bodies.

In summary, the available evidence identifies α-synuclein as a marker of the PD/DLB process but do not prove that it has a causal role. The evidence that it does comes from a variety of human genetic studies.

α-Synuclein can cause PD

A key discovery in understanding PD was the report that an A53T mutation in the α-synuclein gene was causal for dominantly inherited disease [44]. This was the first clear report that a Mendelian gene could be a cause of PD, which to that point had been thought of as a non-genetic disease. It is interesting that the first mutation found was A53T, i.e. a reversion of the human Alanine to the ancestral Threonine found in rodents and many other species. Since then, two other point mutations, A30P [45] and E46K [46], have been reported in different families. It is also important that while many cases are reported to have a phenotype of ‘PD’, in fact several patients in the A53T and E46K [46] families have a more diffuse involvement of synuclein deposition [47, 48] and clinical features that presumably result from this degree of involvement of non-dopaminergic systems [49].

A second group of important cases have multiplications of the normal wild type allele of SNCA, the gene that encodes for the α-synuclein protein. Cases with SNCA duplication have a brainstem-predominant PD phenotype [50], while cases with a triplication have a Lewy body disease that again involves several brain regions [51, 52]. Measurements of protein levels in triplication show the predicted doubling of α-synuclein in blood as well as increased levels and deposition of the protein in the cerebral cortex where Lewy bodies are found [21]. Therefore, even without sequence variants, α-synuclein dosage can be causal for Lewy body disease.

A third piece of genetic evidence comes from the reports common variants around the α-synuclein gene are associated with lifetime risk of sporadic PD. Both the promoter region, specifically the Rep1 polymorphic repeat [53], and polymorphisms towards the 3′ end of the gene are associated with PD [54]. Although it is not known specifically how these risk variants influence lifetime incidence of PD, it seems likely that they increase α-synuclein protein levels in the brain.

Collectively, the human genetic data strongly support a causal role for α-synuclein in PD/DLBD. Whether Lewy bodies are causal or consequential is less clear, but they do support the idea that α-synuclein represents an important link between sporadic and inherited PD. The various lines of evidence identify α-synuclein as a potentially toxic protein, fulfilling the requirements of a causative agent in PD [55]. The question now is how, and in what context, is α-synuclein toxic, and can we do anything about it?

Where and when is α-synuclein toxic?

Given that cell loss is a major event in human PD, combined with the evidence that α-synuclein plays a causal role in disease, it is reasonable to infer that α-synuclein is toxic to human neurons. The time course is likely to be protracted, with the most likely explanation that there is asynchronous cell death that results in a slow depletion of the populations of relatively vulnerable neurons. However, it is not possible to watch cells die in the human brain and so we have to turn to experimental models to confirm or refute the idea that α-synuclein is toxic.

Yeast models are probably the simplest system used to show that expression of human α-synuclein evokes toxic events. In growing and stationary phase cultures, increased expression of α-synuclein limits cell growth [56, 57, 58, 59, 60, 61, 62, 63, 64, 65]. These experiments are extraordinarily useful in defining pathways that underpin the toxic effects of the protein. α-Synuclein toxicity has also been demonstrated in Drosophila, where dopaminergic neuron cell loss has been reported [66, 67, 68, 69, 70, 71, 72, 73], although this result is a little controversial [74] and the effects are modest. The worm C. elegans can also be used to show that α-synuclein can damage dopamine neurons in an intact, in vivo, setting [75, 76, 77, 78, 79, 80]. What links these three model systems is that they all show a detrimental effect of expression of α-synuclein in organisms where the protein is not normally present. One reading of this data is that, at least in terms of toxicity occurring over days to weeks, the normal function of the protein is probably not relevant.

A situation where α-synuclein is normally present is in mammalian cell culture models. Two commonly used systems are primary neurons, including dopaminergic cultures of the ventral midbrain, or neuroblastoma derived cell lines. Experiments showing the most substantial effects of α-synuclein include those where the protein is transiently expressed, e.g. from viral vectors [81, 82, 83, 84, 85, 86], or expression is controlled from an inducible promoter system [87, 88, 89], although some authors have reported a lack of toxicity in similar circumstances [90]. In midbrain cultures, toxicity is higher for dopamine neurons than other cells [81], which may be relevant to the relative vulnerability of nigral neurons in PD. Some experiments show nicely that the difference between wild type and mutant protein is really a matter of dose and that at increasing expression levels, the normal protein becomes as toxic as the dominant mutants [89].

Although potentially useful in for understanding mechanisms, these cell-based models are taken out of theirin vivo context and tend to show cell loss over a few days, compared to the predicted years of progress in the disease. A more intact approach is to express α-synuclein using transgenic technology in various parts of the mouse CNS. Some of these models show toxicity, particularly in the spinal cord, but nigral cell loss is absent or modest [91, 92, 93, 94, 95, 96, 97]. Several models do show accumulation and insolubility of α-synuclein [e.g., [36, 91, 93, 98]], although whether true Lewy bodies are formed is uncertain. Therefore, most mouse models reported to date are better for understanding α-synuclein deposition than frank cellular toxicity. Why this is the case is unclear, but it is interesting that crossing transgenic models with mouse α-synuclein knockouts exacerbates phenotypes [99, 100, 101], suggesting that the presence of the murine protein limits damage in some undefined way. The lack of an ideal PD mouse model that more completely captures the human phenotype limits our current studies of α-synuclein toxicity. Though a goal worth pursuing, creation of such an ideal mouse model may be very challenging given the limitations of mouse lifespan and differences in physiology between mice and humans.

An alternate approach to traditional transgenics is to use viral vectors to deliver α-synuclein directly to the substantia nigra in mice [102], rats [103, 104, 105, 106] or non-human primates [107, 108, 109]. In these approaches, a significant cell loss is noted along with deposition of α-synuclein protein. The extent of cell loss is less dramatic than in human PD and behavioral effects are similarly modest. However, the critical observation here is that α-synuclein can induce toxicity in vivo using vertebrate organisms, with a time course of several weeks, allowing for some dissection of mechanism.

Taken together, all of this evidence suggests that α-synuclein can induce toxicity in a variety of contexts, from simple organisms to dopamine neurons in the primate substantia nigra. It is less clear whether all of these situations are directly relevant to the human disease, where cell loss is probably more protracted, but as a practical matter such models at least afford an opportunity to examine mechanism(s) by which α-synuclein triggers neuronal death.

Why is α-synuclein toxic?

Some of the above model systems have been used to probe the mechanism(s) by which α-synuclein causes cell death. These can generally be sorted into aspects of the protein itself effects of the protein to the biological system (see figure 1). Appendix 1 highlights some of the key observations related to this critical question.

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Figure 1

Events in α-synuclein toxicity. The central panel shows the major pathway for protein aggregation. Monomeric α-synuclein is natively unfolded in solution but can also bind to membranes in an α-helical form. It seems likely that these two species exist in equilibrium within the cell, although this is unproven. From in vitro work, it is clear that unfolded monomer can aggregate first into small oligomeric species that can be stabilized by β-sheet-like interactions and then into higher molecular weight insoluble fibrils. In a cellular context, there is some evidence that the presence of lipids can promote oligomer formation: α-synuclein can also form annular, pore-like structures that interact with membranes. The deposition of α-synuclein into pathological structures such as Lewy bodies is probably a late event that occurs in some neurons. On the left hand side are some of the known modifiers of this process. Electrical activity in neurons changes the association of α-synuclein with vesicles and may also stimulate polo-like kinase 2 (PLK2), which has been shown to phosphorylate α-synuclein at Ser129. Other kinases have also been proposed to be involved. As well as phosphorylation, truncation through proteases such as calpains, and nitration, probably through nitric oxide (NO) or other reactive nitrogen species that are present during inflammation, all modify synuclein such that it has a higher tendency to aggregate. The addition of ubiquitin (shown as a black spot) to Lewy bodies is probably a secondary process to deposition. On the right are some of the proposed cellular targets for α-synuclein mediated toxicity, which include (from top to bottom) ER-golgi transport, synaptic vesicles, mitochondria and lysosomes and other proteolytic machinery. In each of these cases, it is proposed that α-synuclein has detrimental effects, listed below each arrow, although at this time it is not clear if any of these are either necessary or sufficient for toxicity in neurons.

Aspects of protein chemistry of α-synuclein and toxicity

α-Synuclein has a strong tendency to self-associate in vitro [110, 111], and so a prime candidate for the underlying driving force for toxicity is the formation of aggregated species. One of the important questions about this idea is which species are present in cells/tissues. Oligomeric species can be isolated from cells [112,113, 114] and from human [21] and mouse (both wild type and α-synuclein transgenic) brain [115]. In both cells and brain, oligomers are particularly found in membrane-enriched fractions [112, 115], suggesting a possible influence of the lipid environment on oligomer formation. Higher molecular weight forms have also been found in some models [116], especially after oxidative stress [117] or exposure to inflammatory triggers in mice [100]. Deposited α-synuclein immunoreactivity has been seen in transgenic [91, 92, 93, 94, 95, 96, 97] or viral models [102, 103, 104, 105, 106, 107, 108, 109]. However, the observation of aggregated α-synuclein by and of itself does not prove that aggregation is important; as discussed for Lewy bodies, all this proves is that deposition occurs, not that it is causal.

Some recent studies have attempted to answer this question, mainly using cell-based approaches. For example, some oligomeric forms of α-synuclein trigger calcium entry and toxicity in SY5Y cells [118]. Interestingly, different species show differential toxicity, suggesting that not all oligomers are created equal. However, the nature of this experiment is to add α-synuclein to the outside of the cell, which may or may not be relevant to the pathophysiological situation. As α-synuclein is intracellular, it seems more likely that the protein would form aggregate inside cells. The presence of fibrils in Lewy bodies would support this contention. However, α-synuclein can end up in the extracellular media [119] and it is possible that the conditions for aggregation might be more suitable in a milieu free of cells. The relevance of extracellular α-synuclein is an important question, raised also by the observation of Lewy bodies in grafted neurons [41, 42] and the attendant hypothesis of ‘host to graft transmission’.

Some studies have attempted to address whether intracellular aggregates of α-synuclein contribute to toxicity. For example, several imaging techniques shown that, in the context of a living cell, α-synuclein can form small oligomers, likely in an antiparallel configuration [114, 120] and such oligomers can be associated with cell toxicity.

These approaches have been used to show that overexpression of heat shock proteins (Hsps) can mitigate both oligomer formation and toxicity [114, 120, 121]. In vivo, Hsps can prevent toxic effects of α-synuclein in yeast [59] and in flies [67]. Whether these studies constitute formal proof that aggregation is required for toxicity is unclear as there are other theoretical interpretations of the data. For example, a formal possibility is that monomeric α-synuclein is toxic and, thus, any protein binding the protein directly could limit toxicity. It should be stated that the mechanism(s) by which monomers of α-synuclein could be toxic are relatively unexplored but, equally, there is an absence of proof that aggregation is absolutely required for toxicity. Alternatively, Hsps could be limiting a detrimental event downstream of the initial aggregation and thus may neither represent evidence for or against the role of aggregation in α-synuclein toxicity. Interestingly, Hsp expression in the fly model decreases neuronal toxicity without any change in the number of α-synuclein positive inclusions [67].

Overall, these considerations show that α-synuclein is capable of protein aggregation and can be deposited into inclusion bodies of various forms in vivo, but that there is insufficient evidence that aggregation or deposition is either necessary or sufficient for toxicity. In fact, several lines of evidence show that toxicity can be dissociated from deposition, including; the observation in cells of toxicity without deposition in some models [81]; differential effects on toxicity and inclusions of various manipulations of α-synuclein in fly models [66, 67]; and deposition of α-synuclein without clear toxic effects in some mouse models [e.g., [36]]. A key challenge for the field, therefore, is to understand whether protein aggregation is at all relevant for the toxic effects of α-synuclein. One way to potentially address this is to isolate various aggregated species of the protein and to express them within a neuron. This might be extraordinarily difficult from a technical standpoint and there is always possibility that the small aggregates would seed larger ones may confound interpretation. Another potential approach would be to develop reagents that limit the biological availability of specific aggregated species and use these to probe which agents are toxic in intact cells. As an example, recombinant single chain Fv antibody fragments against aggregated α-synuclein have been described [122,123] that might be helpful.

α-Synuclein has many additional properties as well as the tendency to aggregate. Some of the post-translational modifications that have been reported have also been explored as possible mediators of toxicity. For example, antibodies against α-synuclein phosphorylated at Ser129 are very good at identifying Lewy pathology in the human brain [38], suggesting either that Ser129 phosphorylation is a causal event for deposition or represents a common modification of the protein after it is deposited. Several groups have therefore made versions of α-synuclein that cannot be modified at this residue (S129A) or pseudo-phosphorylation mimics (S129D, S129E) and determined the toxic effects of expression. In Drosophila models, S129A is less toxic but has an increased tendency to form inclusion bodies compared to wild type protein [66]. The S129D phosphomimic has the opposite effect, i.e. increased toxicity but fewer inclusions. In contrast, similar experiments using viral overexpression in rats show the opposite result, namely that S129A greatly increases the toxic effects of expression [124]. In mammalian cell culture, S129A has a diminished tendency to form inclusion bodies [125].

At first glance, these results seem to suggest that the behavior of α-synuclein as it relates to toxicity is opposite in mammals compared to invertebrates where, it is important to note, the protein is not normally present. However, interpretation is complicated by several considerations. First, the expression levels of α-synuclein are critical for toxicity, which is shown by the human case where a difference in protein levels is 2-fold in the triplication cases and 1.5-fold in the duplication cases. Second, recent data suggests that the phosphomimic S129D/E α-synuclein variants have different biophysical properties compared to authentically phosphorylated wild type protein [126]. Overall, these considerations raise some important caveats about comparison of properties of α-synuclein in terms of concentration-dependent behaviors of the protein such as aggregation and toxicity.

One alternate approach to understand α-synuclein phosphorylation is to identify the kinase that mediates the phosphotransfer event. Casein kinase II and GRK2/5 have been shown to phosphorylate α-synuclein in vitro or in cells and work in yeast [64] and flies [66] respectively shows that they are at least active in vivo. More recently, the polo-like kinase family, specifically PLK2, have been shown to be active both in vitro and in vivo in generating pS129 α-synuclein [127]. What is interesting about PLK2 is that it is known to respond to neuronal activity [128], suggesting a possible link between neuronal phenotype and α-synuclein toxicity. However, it is not yet known in PLK2 inhibitors or gene knockout will limit the toxic effects of α-synuclein in vivo. Such experiments are feasible in several species as PLK2 homologues are present in mice and flies, and there is at least one polo kinase in yeast.

There are a number of other modifications of α-synuclein that have been reported and some of these are found more often in pathological circumstances than under normal conditions, such as nitration or truncation. Truncation of α-synuclein is associated with a higher tendency for aggregation [129, 130, 131]. Transgenic mice expressing truncated α-synuclein have substantial cell loss [101] although in at least one line, this is a developmental and not degenerative phenotype [132]. Again, because the window for toxicity is quite narrow, comparison between different lines is difficult. One question that arises for truncation is where such species are generated. α-Synuclein is predominantly degraded by lysosomal pathways [133, 134], including chaperone-mediated autophagy [135], and the lysosomal cathepsins are important in proteolysis. Therefore, some truncated species are found in the lysosomes and it seems unlikely that they would cause damage to the cell. However, α-synuclein is also a substrate for cytoplasmic calpains [136, 137, 138, 139], which are therefore more likely to generate cytoplasmic toxic truncated species. Some detail is therefore needed to prove which truncated species mediate toxicity, if any of them in fact do.

Oxidative stress, including the neurotransmitter dopamine, has been linked to increased α-synuclein aggregation [89, 140]. Dopamine itself may contribute to the toxic effects of α-synuclein in vitro [89], although such a mechanism cannot explain why non-dopaminergic neurons die early in the disease process. α-Synuclein expression can enhance sensitivity to oxidative and nitrative stressors [141, 142], although it can also be protective in some situations [143]. In most of these situations, the role of aggregation is unclear.

In summary, α-synuclein has properties, including the potential for aggregation and post-translational modifications, which may influence its toxic effects. Whether these are required for toxicity is unclear, and some results still need to be resolved, for example for the work on S129 phosphorylation. However, there is a larger question, which is: what effects synuclein has on neurons that are responsible for its toxic effects?

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Mediators of α-synuclein toxicity in biological systems

Some of the relevant data from cellular systems has been reviewed previously [144] and will be discussed here in the context of examples across multiple models.

Presumably, α-synuclein might interact with other biomolecules to mediate toxicity. Because α-synuclein can associate with lipids, membranes are one possible target. In vitro, α-synuclein can form pore-like structures [145, 146], and annular rings of synuclein have been isolated from the brains of patients with multiple system atrophy, a synucleinopathy [147]. Cells expressing α-synuclein have increased cation permeability [148] and vesicles prepared from cultured cells or isolated from the adrenal medulla show leakage of catecholamines [149]. These events may be consistent with the formation of non-specific pores or similar structures at the plasma membrane or at a vesicle surface.

Because α-synuclein binds synaptic vesicles, it is possible that synaptic transmission would be directly or indirectly a target of synuclein toxicity. One example of this comes from work showing that A30P α-synuclein alters exocytosis of catecholamine containing vesicles in primary cells and in chromaffin cells [150]. The effect here is probably at a late stage of the exocytosis, before vesicle membrane fusion [150].

Further evidence for an effect of α-synuclein on vesicle function that may mediate toxicity comes from suppressor screens in yeast [63]. In the same organism, such defects can be localized to a block in endoplasmic reticulum (ER)-golgi vesicular trafficking [151]. Supporting this idea, there is evidence of ER stress [87] and golgi fragmentation [152] in mammalian cell systems.

Overexpression of Rab1, a GTPase that influences vesicle dynamics, was able to at least partially rescue the toxic effects of α-synuclein in yeast, worms and in mammalian cells [151]. Therefore, some of the toxic effects of α-synuclein that are conserved across species involve damage to vesicular transport, which might express itself as damage to presynaptic vesicle release in a neuron.

There are also suggestions that other membranous organelles are affected by α-synuclein, including mitochondria [87, 88, 153]. Recent data suggests that a portion of α-synuclein can localize to mitochondria, at least under some conditions [154, 155, 156, 157]. Supporting this are observations that α-synuclein expression increases cellular organismal sensitivity to rotenone, a mitochondrial complex I inhibitor [78, 158]. Furthermore, intact mitochondrial function is required for a-synuclein toxicity in a yeast model, although it should also be noted that removal of mitochondria is also quite damaging in the same context [57]. The mechanism by which α-synuclein interacts with and causes damage to mitochondria is not fully resolved and, given the central role of mitochondria in apoptotic pathways, perhaps such effects are secondary to the induction of apoptosis. Increased levels of α-synuclein are reported to trigger apoptosis in various cell types [159, 160, 161]. Several apoptotic markers are also seen in yeast models of synuclein toxicity [59]. α-Synuclein toxicity can be rescued by caspase inhibitors or knock down of caspase-12 [87]. Activation of caspase-3 has been reported in transgenic mice [162] caspase-9 has been reported in viral models in mice [102] and rats [106]. However, these studies show only a few caspase positive cells, and so whether apoptosis is the only way in which cells expressing α-synuclein die remains unclear.

α-Synuclein can bind to the membranes of lysosomes [135] and inhibit lysosomal function [163] and chaperone-mediated autophagy [135]. Recent results suggest that CMA is implicated in the regulation of the transcription factor MEF2D and that this can be disrupted by expression of α-synuclein, leading to neuronal death [164]. As another example of misregulated protein turnover, α-synuclein (and specifically α-synuclein oligomers) can also inhibit the proteasome [81, 88, 163, 165, 166, 167], although it is not clear if the predicted altered turnover of proteasome substrates occurs in vivo [168].

The general principle is that multiple systems can be affected by α-synuclein expression and that if there is a common theme between them, it is likely to be that α-synuclein can binds lipids. Several lines of evidence suggest that lipid binding can promote the formation of oligomers [115, 145, 169]. Therefore, this interpretation links a primary protein abnormality to cellular targets of the protein. As discussed elsewhere [144], determining which events are truly primary and which are secondary remains a challenge. Although this distinction is an intellectual problem, it may also be relevant for deciding which aspects of cell death to target if we want to limit the disease process in PD.

Potential therapeutic approaches related to α-synuclein toxicity

One of the key questions here is to decide whether to try and target the protein or the process that mediates cellular damage. Both are attractive for different reasons, although both are also difficult (see figure 1 for where these might be utilized and Appendix 2 for the critical next steps).

If there were a pathogenic aggregated form of α-synuclein, then one tactic would be to target that species. If we propose that insoluble fibrils are toxic, then a ‘fibril-buster’ would be the way forward [reviewed in [111]], but if soluble oligomers damage cells then we would want to prevent their formation or encourage their turnover. As discussed above, both fibrils and oligomers can be found in different models and either alone, or both, could be toxic. For oligomers, the situation is more complicated if different oligomeric forms have different toxic properties [118], suggesting that we may need to be careful about which oligomers we target.

Alternatively, we could be agnostic about which species are important and try and decrease all α-synuclein expression. There are reports that increasing autophagy can help clear aggregation-prone proteins, including α-synuclein [170]. Antisense approaches might also be helpful, and have been reported to work in the rat [171] and mouse [172] brain. This approach is predicated on the idea that α-synuclein really is dispensable for CNS function in humans, as it appears to be in the mouse [28, 30], but perhaps even a modest decrease in protein levels would be enough to decrease PD progression.

We might also try to change the modifications of α-synuclein, especially if these are specific for pathogenic forms. For example, example of PLK2 as a kinase for Ser129 [127] may provide a way to test the idea that phosphorylation at this residue is key for pathogenesis, if sufficiently specific kinase inhibitors can be developed. Again, assuming specificity can be achieved, it might be interesting to block other modifications such as truncation or nitrosylation – the latter might be part of the general rubric of anti-inflammatory approaches. However, such approaches would only be helpful if the modification is truly specific for the pathogenic form and makes an active contribution to cellular toxicity, ie is not a bystander in the process.

Finally, we may target one or more of the cellular effects of α-synuclein that are associated with toxicity. This might have the advantage of leaving the protein alone, which may be useful if it turns out that α-synuclein has a specific function in the human brain. The difficulty, of course, is in understanding why the protein is toxic, although the work with Rab1 [151, 173] suggests that this is a tractable problem, at least in principle.

Conclusion

Cell death is a significant part of the pathology of PD. Although the process is a mysterious, the prime suspect for a toxic protein is α-synuclein. Assuming toxicity does indeed result from aberrant forms of the protein, including increased expression of the normal gene, there are two major aspects that might be targeted therapeutically. First, the protein is prone to aggregate and anti-aggregative compounds, or approaches to simply limit net expression levels, may be helpful. Second, there are a number of molecular events that largely revolve around membrane or organelle interactions that may contribute to toxicity, and these too may be targeted therapeutically. Future work should be directed at exploring these possibilities as well as at developing models that have a stronger cell death signal, to more accurately represent the substantive loss of neurons seen in PD.

Appendix 1: key observations

The role of α-synuclein in PD and related disease is highlighted by the convergence of pathological and genetic data. Because part of the pathological phenotype of PD involves cell death of neurons, particularly but not exclusively dopamine neurons in the substantia nigra pars compacta, this suggests that α-synuclein may be a toxic protein. The following key observations have been made in various experimental systems to support this contention:

– In pure in vitro assays, α-synuclein shows a lack of conformational restraint that tends to promote inappropriate aggregation. This can be enhanced by mutation, increasing concentration or any of several protein modifications associated with pathological deposition of the protein in vivo. α-Synuclein can also bind lipids and membranes in vitro

– In a variety of species, expression of α-synuclein can promote toxic events. These include organisms such as yeast, worms and flies, where no α-synuclein homologue is present, suggesting that irrespective of its normal function, the protein can be toxic.

– Data in mammalian cell culture also supports a toxic effect of α-synuclein, particularly to dopaminergic cells. Results in intact in vivo systems are mixed, with toxicity limited to the spinal cord in some transgenic mouse models and modest toxic effects to dopaminergic neurons using viral mediated overexpression in rodents and non-human primates.

– The mechanism(s) involved are currently unclear, but binding to several cellular membranes may contribute to toxic events.

Appendix 2: critical next steps

The following critical issues need to be addressed before our understanding of α-synuclein pathobiology can be applied to therapeutic development:

– We need to better understand normal function of α-synuclein, such that we can assess both what role it might play in toxicity in the mammalian CNS and so we can highlight potential detrimental effects of limiting expression or function of the protein.

– We need to clearly identify which cellular pathways contribute to the pathological effects of the protein. Some great work has been performed in yeast models that highlight interruption of vesicle transport, but it is important now to establish what the analogous process is in neurons and whether this is sufficient to explain α-synuclein toxicity in this system.

– We need to develop models where there is a lesion that better approximates the severity of cell loss seen in human PD. This will allow for a more rigorous test of pathways involved in toxicity as the disease progresses. An accelerated time course would be helpful, and may be necessary, but the pathology should be similar to human PD in that nigral neurons should be affected at some point in the model but not necessarily first or exclusively.

Abbreviations

DLB/DLBD: 

Dementia with Lewy bodies/Diffuse Lewy Body Disease

ER: 

endoplasmic reticulum

L-DOPA: 

3,4-dihydroxy-L-phenylalanine

PD: 

Parkinson disease.

Declarations

Acknowledgements

This research was supported by the Intramural Research Program of the NIH, National Institute on Aging.

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