Alzheimer's Disease - From Fundamental Insights to Light at the End of the Translational Tunnel / Parkinson’s Disease: Genetics, Mechanisms and Therapeutics
More than 200 researchers braved dizzying altitude, howling coyotes, and a fairly brooding mood in light of elusive trial success, to exchange the latest on Alzheimer's and Parkinson’s diseases in joint symposia held March 2-7 in Keystone, Colorado. Between plenary and parallel talks, bustling poster sessions, and the odd foray to the slopes, scientists discussed trial design, genetics, pathology, biomarkers, and cell and protein biology.
Just after the first A4 trial volunteers were screened in San Diego on February 28, study leader ReisaSperling, Brigham and Women's Hospital, Boston, was already laying out her vision for A5, A6, and even A7 trials at a Keystone symposium in Colorado held March 2-7. Called Alzheimer's Disease—From Fundamental Insights to Light at the End of the Translational Tunnel, the meeting ran in parallel with the first-ever Keystone symposium devoted to PD, called Parkinson's Disease: Genetics, Mechanisms, and Therapeutics. A panel on combination therapy generated the most animated discussion at this joint meeting. At issue was whether those future trials will combine two different treatments, as Sperling hopes. The conversation exposed how difficult it might be to test combination therapies in the current climate of negative AD trials. While some academics believe combination trials should begin right away, colleagues in industry are less enthusiastic, seeing all manner of obstacles.
Combination therapy is hardly a new concept in AD. Last year, at the urging of Rusty Katz, who was then at the U.S. Food and Drug Administration, the Washington-based advocacy group ACT-AD and the Critical Path Institute, Tucson, Arizona, hosted a meeting on the topic. The upshot was that the FDA encourages combination trials for AD, and many in the field believe the climate is ripe and the tools are in hand to try (see Feb 2013 news story). At Keystone, Dennis Selkoe, also from Brigham and Women's, convened a panel with Sperling, Ryan Watts from Genentech, San Francisco, and Charles Albright and Michael Ahlijanian of Bristol-Myers Squibb, Wallingford, Connecticut, to gauge support for the idea and discuss how it could be realized. Participants decided to forgo the customary Keystone sojourn to the ski slope, instead packing this mid-afternoon session and peppering it with comments.
Researchers from industry, both on the panel and in the audience, broadly agreed that two or more pharmaceutical companies would willingly collaborate to run such a trial. There are precedents for conducting combination trials, for example in cancer and HIV. "Collaboration is the least of our problems," noted Albright. The issue is not if this can be done, but whether it should be, he said. Albright claimed that because no single agent has yet proven successful in AD, putting two drugs together would amount to a Hail Mary pass: If it failed, it could cripple investment in AD drug development. The risk tolerance of investors in AD has reached an all-time low even for monotherapy, said Albright. "That we might do something even more risky, considering the drug-dosing and safety hurdles that need to be crossed, seems unrealistic," he said. "We need to focus on succeeding with a single agent so that we can spur more investment in this arena, not less."
Watts echoed some of those concerns, conceding that thinking about dose-ranging alone was enough to give him a headache. He reminded the audience that other combination trials, in cancer for example, tended to be conducted on drugs that were first developed individually. "If you see no efficacy unless you combine, then that is a bigger challenge," he said. "Without evidence of clinical efficacy, the risk is extremely high to test two unproven new molecular entities."
Ahlijanian agreed that dosing could be problematic, and cautioned that the safety and tolerability issues may be complex. He said that AD patients can react differently than normal individuals. "Combining drugs that have unknown effects in a patient population may be fraught with danger," he said.
Others worried about regulatory approval and reimbursement decisions that might follow on from combination trials, specifically when trying to determine which of the agents was responsible for any positive effects.
For her part, Sperling considered many of these concerns surmountable. She said the major rationale for a combination trial is that AD represents a combination of pathologies. "We know it is a complex disease, so the idea that targeting a single mechanism will give us a home run is unrealistic," she said. She noted that tackling different forms of Aβ may be as important as targeting Aβ and another entity, such as tau. Sperling argued that combination trials should start now because the length of AD trials means that waiting for each therapy to show efficacy means another decade, and that is too long. She suggested 2 x 2 designs, where drugs can be tested as monotherapies and in combination in the same trial.
Selkoe put it in practical terms. "If we chose solanezumab, which arguably has shown the best effect so far, and decided to go with the safest and most effective dose, would it be irrational to add a BACE inhibitor or an anti-tau antibody?" he asked. Could we envision doing this in mice and in humans in a year?
Watts was even more direct, asking "Merck, are you guys ready to combine your BACE inhibitor with Lilly's antibody?" Following a brief pause, Merck’s Mark Forman—acknowledging that such a decision would be made higher up the pay scale—replied that this seems feasible in animals, but insisted that in the clinic it makes more sense to first see how BACE inhibitors work alone. "We would argue that we have a level of Aβ reduction that is unprecedented and should test [our inhibitor] as a monotherapy," he said. Lilly scientists agreed that combination testing in animals is conceivable in principle.
There remain many issues to iron out, even for preclinical modelling. As in previous discussions of AD combination trials, proponents recommended adaptive trial designs such as those that have been used in cancer trials, while others asked which outcome measures could be used. "To adapt a trial based on cognitive change would take too long," said Sperling. She suggested biomarkers, but Albright noted that none of those have robustly shifted in trials in response to drug. "We have diagnostic and pharmacodynamic markers, but no surrogate markers. Do we have an outcome that we can adapt on?" he asked.
Dennis Dickson, Mayo Clinic, Jacksonville, Florida, advised that the heterogeneity of the disease be taken into account lest trials be underpowered. Selkoe agreed, noting that a substantial portion of participants in the solanezumab trial turned out to be amyloid negative or not have AD, and those who did may have multiple pathologies. Selkoe suggested correlating soluble oligomers in the CSF with disease and phenotyping participants in other ways, including tau imaging. Sperling agreed that endophenotyping would be valuable and said more imaging and biomarker analysis should be part of any new trial. For example, some patients in the A4 trial will undergo brain imaging for tau (see below). Watts said one reason why Genentech opted for the API trial was because its cohort is more homogenous. "We can come out with negative trials time and again, when really the drug has worked for some of those volunteers," he said
Others wondered what drug combination to use and when to begin. Some suggested using a BACE inhibitor and solanezumab. Albright questioned what that would achieve, since both therapies essentially target the same molecule, soluble Aβ. Sperling said she'd combine a BACE inhibitor with an antibody that targets an aggregated form of Aβ, or tau. Watts found that idea compelling, and suggested targeting inflammation in combination with Aβ therapies. He hinted that bi-functional molecules that could be developed as a single drug might circumvent some of the problems associated with combination therapy.
LEARNing from A4
Researchers hope to learn much from upcoming secondary prevention trials. Sperling announced that A4 has now recruited a site in Melbourne, Australia, bringing the total number of centers to 60. Some of the A4 patients will undergo tau PET imaging, a first in any AD trial. The Alzheimer's Association recently announced an $8 million grant for a so-called LEARN arm of A4. LEARN, which stands for Longitudinal Evaluation of Amyloid Risk and Neurodegeneration, is to track cognitive and clinical change in volunteers who test negative in PET scans for brain Aβ accumulation at a screening visit for A4. The grant also funds tau imaging in 100 participants who do have brain amyloid (50 in the treatment arm and 50 in the placebo group), and a further 50 in the amyloid-negative control group. Sperling told Alzforum that which tau ligand will be used has yet to be determined. "We have most experience with T807, but we want to consider all agents," she said. Sperling and colleagues describe the A4 trial in a Focus piece in the March 19 Science Translational Medicine.
A4 will run for three years, at which point Sperling hopes to have A5 in place. For that, she would like to test a BACE inhibitor. For A6, Sperling has previously proposed Combination Therapy in Early AD, aka COMBAT, a trial that could test both a secretase inhibitor and an antibody. She told Alzforum that a combination of an anti-amyloid and anti-tau therapy would be preferable. Subjects in the LEARN trial would be ideally suited to transition into one of these subsequent trials, depending on whether they showed signs of developing Aβ pathology and/or more neurofibrillary tangles. “We have always wondered how we can identify those who are just about to show signs of tau spreading from the medial temporal lobe. Now A4 can perhaps give us that information,” Sperling said.—Tom Fagan.
Blockage of the lysosomal system that degrades unwanted protein has emerged as a common theme in neurodegenerative diseases. At "Parkinson’s Disease: Genetics, Mechanisms, and Therapeutics," a meeting held 2-7 March in Keystone, Colorado, researchers proposed ways to revive that process, including kicking protein trafficking to the lysosome into high gear. The meeting built on the connection between the toxicity of α-synuclein, a hallmark of Parkinson's pathology, and GBA1, a genetic risk factor for the disease. GBA encodes the lysosomal lipid-degrading enzyme glucocerebrosidase (GCase). At Keystone, researchers reported that α-synuclein blocks folding and trafficking of the enzyme to that organelle. They presented small-molecule chaperones that can refold GCase and deliver it to lysosomes, where it could potentially benefit PD patients.
“If you have a version of GCase that’s not folded properly but is still made in the neuron, then the refolding idea is an attractive one,” commented Mark Cookson of the National Institutes of Health. “GBA1 is a genuine PD risk factor for significant numbers of people, so even if you could only treat GBA1-mutation-associated PD, that would be a huge hit.” Because α-synuclein inclusions play a role in other neurodegenerative diseases as well, Keystone attendees proposed that the therapy could be more broadly applied, perhaps even to Alzheimer's.
Mutations in GBA1 also cause Gaucher’s disease (GD), which is characterized by a buildup of lipids in lysosomes. Some forms of GD cause neurodegeneration in the brain (see Jan 2014 news story). The homozygous mutations that trigger GD are rare, but heterozygous mutations in GBA1 are common and increase a person’s chances of developing Parkinson’s disease and dementia with Lewy bodies (see Apr 2013 news story and PDGene).
How GBA1 mutations influence these synucleinopathies remains a hot topic. In 2011, researchers from the lab of Dimitri Krainc, then at Massachusetts General Hospital, Boston, proposed that GCase and α-synuclein form a destructive feedback loop in which the accumulation of the GCase substrate glucosylceramide stabilizes α-synuclein oligomers, which in turn inhibit the activity of the enzyme.
At Keystone, Krainc, now at Northwestern University, Chicago, suggested that the toxic loop may roll in reverse: Accumulation of α-synuclein can dampen GCase activity, increasing glucosylceramide. To arrive at this conclusion, Joseph Mazzulli, who was a postdoc with Krainc but now runs his own lab at Northwestern, nurtured midbrain neurons derived from induced pluripotent stem cells (iPSCs) made from a PD patient who carried two extra copies of the a-synuclein gene. Mazzulli grew the neurons for more than a year, a feat that awed researchers at the meeting (see image below). As the cells aged, the neurons with the α-synuclein triplication started ramping up production of lysosomes. However, after about 200 days in culture, “that compensation started to fail,” Krainc said. Despite the increase in lysosomal biogenesis, α-synuclein gradually accumulated in the cells, and the level of the protein was “striking” by day 200, he said. After 420 days the cells were still viable, however, the activity of GCase was dramatically lower than in neurons derived from normal iPSCs. The researchers observed an accumulation of GCase in the endoplasmic reticulum (ER) fraction, suggesting that trafficking of the enzyme from the ER through the Golgi was blocked. Interestingly, the α-synuclein accumulation also seemed to hold up other enzymes that traffic through the Golgi pathway, such as β-galactosidase.
Synuclein Surge:
Compared with control cells (top panel), midbrain neurons (green and blue) derived from a PD patient who had two extra copies of the α-synuclein gene (bottom panels) showed signs of α-synuclein accumulation (red) after 60 days in culture. [Image courtesy of Dimitri Krainc.]
Krainc reasoned that “opening up lanes” in the ER-to-Golgi pathway might relieve the congestion. Indeed, when the researchers overexpressed Rab1a, a GTPase known to promote ER-to-Golgi trafficking, α-synuclein levels in the neurons dropped, and GCase activity was restored. The researchers achieved a similar result by dosing the cells with a small-molecule chaperone of GCase (Rogers et al., 2013). The molecule helps GCase fold properly and ferries the enzyme through the vesicular pathway into the lysosome, where it can do its job, Krainc said. Because the chaperone approach works to enhance the function of both wild-type and mutated forms of GCase, companies are pursuing similar molecules as potential therapies for PD and GD.
Several researchers in the audience praised the work, but some questioned whether this approach could be applied to patients with sporadic PD, who do not all have the same amount of α-synuclein accumulation. Others wondered whether the approach could work for other neurodegenerative diseases marked by protein toxicity. Krainc cautioned that the GBA1 target was, so far, mechanistically linked only to α-synuclein accumulation.
“It is very hard to know how well the mechanisms will generalize,” Cookson told Alzforum. “But α-synuclein has always been thought of as a difficult target, so maybe targeting GBA1 is a way into α-synuclein biology and α-synuclein-based therapy.”
Pfizer is one company interested in targeting PD through GBA1. Zdenek Berger from the company's Cambridge, Massachusetts, lab reported that compounds identified in the same screen that Krainc used for his studies activated GCase in human brain extracts. Pfizer is pursuing GCase as a target for several neurologic diseases, Berger told Alzforum.
Patrick Lewis of the University of Reading in England commented that while the link between GBA1 and synucleinopathies is gaining traction, it is important to remember that not everyone with a GBA1 mutation develops PD. “It’s a risk factor, but not a directly causative mutation,” Lewis said. “That makes the translation more difficult.” Nevertheless, Lewis acknowledged that another genetic link strongly implicates lysosomal dysfunction in PD. Mutations in the lysosomal transport gene ATP13A2 lead to early onset parkinsonism and α-synuclein accumulation (see Usenovic et al., 2012, and Tsunemi et al., 2013).
The Keystone meeting featured more data that meshed with α-synuclein’s ability to waylay GCase in the ER. For example, Vikram Khurana of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, suggested that ramping up ER-associated degradation (ERAD), which disposes of misfolded proteins by trafficking them out of the ER, can overcome α-synuclein toxicity as well. Khurana’s recent yeast screen identified Hrd1, an E3 ubiquitin ligase that promotes ERAD, as a suppressor of α-synuclein toxicity. Overexpressing Hrd1 in iPSC-derived neurons from a PD patient with the A53T α-synuclein mutation reduced both α-synuclein accumulation and the retention of GCase in the ER (see Oct 2013 news story). Further, Khurana reported reduced α-synuclein toxicity and less build-up of GCase in the ER as a result of treatment with N-aryl benzimidazole (known as NAB2), a small molecule identified in a yeast screen as a promoter of endosomal trafficking, These results resemble Krainc’s on Rab1a overexpression or treatment with GCase chaperones. Overall, researchers from several labs presented a handful of approaches using different ways of increasing ER-mediated or lysosomally mediated protein degradation, all of which alleviated cellular phenotoypes of PD pathology.
“If the trafficking of GBA1 is perturbed by α-synuclein, then we’re hoping that NAB2 could be a compound that would reverse that trafficking defect,” Khurana said. “Because α-synuclein may perturb trafficking of many different kinds of protein, a compound like NAB2 might be able to rescue multiple defects at once.”
Khurana will conduct more comprehensive yeast screens to identify genes that rescue or exacerbate α-synuclein toxicity either alone or together. From that, he plans to build genetic interaction maps that could help probe human iPSC-derived neurons for what he calls “druggable nodes,” or pathways implicated in disease pathology.
Besides GCase, Khurana also found that nicastrin, a component of γ-secretase, accumulates in the ER of PD neurons. Whether this connects AD to synucleinopathy remains to be shown, but it does hint that protein trafficking defects may play a broader role in neurodegenerative disease, said Khurana.
Along that vein, Hui Zheng of Baylor College of Medicine in Houston implicated a transcription factor that controls autophagy in cleaning up another AD pathology. Zheng found that overexpression of the factor reduced the presence of neurofibrillary tangles and rescued neurodegenerative defects, including neuronal loss, deficits in synaptic plasticity, and declines in learning and memory as measured by the Morris water-maze challenge, in Tg4510 mice expressing a mutant human tau gene. Zheng, who co-organized the conference, suggested that the factor clears tau via a two-pronged approach: by directly turning on lysosomal genes, and by boosting the expression of PTEN, a protein that promotes autophagy.
Said Khurana, “Lysosomal dysfunction has emerged as a theme encompassing different neurodegenerative diseases, but the mechanisms that perturb endosomal trafficking and lysosomal function may be highly distinct between the diseases.”—Jessica Shugart
Prodromal Initiative to Identify Biomarkers for Parkinson’s
The hunt continues for harbingers of Parkinson’s disease that may one day allow patients to be treated before symptoms take hold. A Keystone conference called “Parkinson’s Disease: Genetics, Mechanisms, and Therapeutics,” held 2-7 March in Keystone, Colorado, revealed that progress has been slow. No new reliable markers have surfaced, though researchers have recruited presymptomatic individuals considered at high risk for the disease to search for preclinical markers in an early version of the Parkinson’s Progression Markers Initiative (PPMI). Others continue to pursue diagnostic blood tests, which have proven hard to find for many neurodegenerative diseases.
“We need PD biomarkers to facilitate new models for drug design that would help us understand the earliest pathology in disease,” said Kenneth Marek of the Institute for Neurodegenerative Disorders in New Haven, Connecticut. “With these biomarkers, ultimately, we could identify groups prior to the onset of symptoms and treat them with drugs that could prevent disease.” Marek also noted that the biomarkers could serve as key measures of disease modification in response to therapy.
Marek kicked off the biomarkers session at Keystone by describing an expansion of PPMI. Started three years ago, the initiative spans 24 study sites in the United States, Europe, and Israel. The study’s 400 newly diagnosed PD patients and 200 controls undergo extensive brain imaging and regularly donate CSF and blood samples. The hope is that biological monitoring will reveal biomarkers that correlate with disease progression. Early data from the cohort revealed that the PD patients had lower levels of α-synuclein, tau, and phosphorylated tau in the CSF (see Sep 2013 news story). While the patients enrolled in PPMI are in the earliest stages of PD, Marek is in search of prodromal markers. “We wanted to move back in time and see if we could enroll asymptomatic people at risk for disease,” Marek told Alzforum. “The challenge is, how do you find these people?”
Marek laid out his strategy for adding another P, for prodromal, to the PPMI cohort. To find patients who fit the bill, he is taking advantage of two early symptoms that often precede the onset of PD: loss of the sense of smell and a sleep disorder called REM Behavior Disorder (RBD), in which people physically act out dreams, kicking and sometimes even falling out of bed (see Aug and Sep 2010 news stories). Recruited via scratch-and-sniff tests sent in the mail and outreach events, respectively, patients positive for either of these predictors will undergo brain imaging for deficits in dopamine transporter (DAT). Using these three combined measures, Marek hopes to recruit patients who are likely to develop PD symptoms within two to five years. The prodromal PPMI is slated to recruit 100 such patients, and so far has found about 20, Marek said.
Another cohort to be included in PPPMI is people who carry mutations in LRRK2 and α-synuclein (SNCA). Because fewer than 2 percent of PD patients have the most common LRRK2 mutation, G2019S, Marek and colleagues are reaching out to the Ashkenazi Jewish population, in whom the frequency is 15 percent of those with the disease. Focusing the search to areas such as Boca Raton, Florida, where many older Ashkenazi Jews live, has boosted recruitment, Marek said. For the even rarer SNCA mutations, Marek is looking to Greek and Southern Italian populations, which harbor higher rates.
Other researchers anticipated future discoveries to come out of the prodromal PPMI cohort. “The ‘triple-P MI’ is very exciting,” said Alice Chen-Plotkin of the University of Pennsylvania in Philadelphia. Once the cohort is fully assembled, she hopes to test blood samples for biomarker candidates she has identified in discovery screens.
Chen-Plotkin believes she can find PD biomarkers in the blood, a goal other researchers consider lofty. Most think the CSF is likelier to contain such markers. However, Chen-Plotkin said that barriers to CSF collection still exist in clinical settings. Trained staff must collect the sample, and some patients are uncomfortable with undergoing lumbar punctures, especially on a regular basis. “Developing a blood-based marker may be harder in some ways than finding one in CSF, but if you are successful, the ability to translate it widely and quickly is so high that it’s worth looking,” said Chen-Plotkin, who works as both a researcher and a physician.
Chen-Plotkin employed multiplex assays to screen the blood of PD patients and controls for more than 100 different proteins, looking for associations with other factors related to disease progression, such as the age of onset or the severity of cognitive decline. She had previously found that PD patients with low cognitive scores tended to have lower levels of epidermal growth factor (EGF) in their blood, and that patients with lower levels of ApoA1 had an earlier age of onset and more severe symptoms (see Qiang et al., 2013, and Chen-Plotkin et al., 2011). She said she and others have since replicated these results in different cohorts. Chen-Plotkin’s group screened the blood of patients enrolled in the Parkinson’s Associated Risk study (PARS), a precursor to the prodromal PPMI study. In this cohort, she found that a higher dopaminergic transporter signal on DAT scans correlated with higher plasma levels of ApoA1. Interestingly, a Taiwanese and U.S. study reported lower incidence of PD in people taking cholesterol-lowering statins, which can raise ApoA1 levels (see Lee et al., 2013, and Gao et al., 2012). If studies were to show that low levels of ApoA1 hasten the onset of PD, it would become possible to treat patients with ApoA1-boosting drugs to stave off disease, but such a strategy is a still a long way down the road, Chen-Plotkin said.
Other researchers cautioned that finding biomarkers in blood would be difficult. “Blood-based markers have been elusive,” said Thomas Montine of the University of Washington, Seattle, “Lots of people have tried, but it looks like Chen-Plotkin is getting some traction.
In his talk, Montine focused on markers of oxidative damage. For decades, researchers have explored links between oxidative stress and neurodegeneration, but they have not answered the question of whether oxidative stress precedes disease or hastens its arrival. Montine and colleagues screened CSF samples from 320 healthy people aged 21 to 100 for the oxidative damage marker isoprostane. Levels of the protein increased with age, but it correlated more strongly with lifestyle factors such as smoking, body-mass index, and diet, than aging alone. Interestingly, people who ate a low-fat diet for a month managed to reduce their CSF levels of isoprostane.
Attendees wondered whether isoprostane was an early indicator of latent disease, or just a marker of aging. “We can’t yet say how important this type of injury is to age-related susceptibility to AD or PD,” Montine told Alzforum. “But our data suggests that there are lifestyle changes that are associated with having less of this type of injury. Presumably that’s a good thing, and it could make the brain less permissive to these diseases.”—Jessica Shugart
Qiang JK, Wong YC, Siderowf A, Hurtig HI, Xie SX, Lee VM, Trojanowski JQ, Yearout D, B Leverenz J, Montine TJ, Stern M, Mendick S, Jennings D, Zabetian C, Marek K, Chen-Plotkin AS.
Plasma apolipoprotein A1 as a biomarker for Parkinson disease.
Ann Neurol. 2013 Jul;74(1):119-27. Epub 2013 Aug 6
PubMed.
Researchers Build on GWAS to Parse Genetic Players in AD and PD
The train of discovery powered by genome-wide association studies (GWAS) is losing steam, so researchers are turning to a different experimental fuel to drive progress on the genetics of Alzheimer's and Parkinson's diseases. The sense of slowing GWAS returns and accelerating alternatives were recurring themes at joint Keystone meetings called Alzheimer's Disease—From Fundamental Insights to Light at the End of the Translational Tunnel and Parkinson’s Disease: Genetics, Mechanisms and Therapeutics, Symposia. Both were held 2-7 March in Keystone, Colorado.
Andrew Singleton of the National Institute on Aging, Bethesda, Maryland, set the tone with his estimation that one-third of PD risk is genetically heritable, yet only about 10 percent of the responsible genes are identified (see Keller et al., 2012). GWAS have unearthed common variants that pose low risk, Singleton said, but fall short when it comes to discovering rare variants that pose higher risks. Furthermore, for each locus that pops up in a GWAS, researchers have to parse the one among dozens of genes nearby that is affected by the variation.
Reinforcing Singleton, Thomas Gasser of the University of Tübingen, Germany, said that as GWAS continue to grow larger, researchers reap smaller rewards. Gasser presented unpublished findings from a recent GWAS doozy, which included nearly 14,000 PD patients. The study yielded 28 PD-associated loci, six of which were new, he said. “Larger sample sizes yield more hits, but at some point it starts to level off,” Gasser noted, pointing to a plateauing graph of GWAS hits versus sample size. “It may not make much sense to keep going higher.” Furthermore, all 28 loci Gasser identified pose only a combined 3.3-fold elevated risk for PD—a level he called “sobering.”
AD geneticists may have plucked all the low-hanging fruit, as well. GWAS by Gerard Schellenberg, University of Pennsylvania, Philadelphia, and colleagues, which together comprised more than 70,000 people, turned up 22 AD risk variants of which 11 were new (see Oct 2013 news story). However, Schellenberg said their GWAS has not yet outlived its usefulness; it can be combined with other techniques that increase power to uncover elusive genotypes. "We rely on imputing to determine genotypes," he told Alzforum. Geneticists use imputation, which is based on the knowledge that certain sequences are usually co-inherited, to fill in missing sequence data. Researchers have incorporated data from the 1,000 Genome Project for GWAS imputation, but this dataset contains sequencing information from 14 different ethnic backgrounds, meaning any one population is represented only by a few hundred sequences or less (see Nov 2012 news story). "Once we have 25,000 genomes sequenced, we can better impute, and that will help us find rare variants," said Schellenberg.
Nevertheless, Richard Mayeux, Columbia University, New York, noted that the field is now leaning more on whole-genome and whole-exome sequencing. For example, last year exome sequencing identified variants in the phospholipase D3 gene that double the risk for AD (see Dec 2013 news story). Mayeux and Schellenberg are on the steering committee for the Alzheimer's Disease Sequencing Project (see Dec 2013 news story). It will sequence the genomes of 566 people in 111 families plus exomes in 11,000 other individuals, including 500 cases and the same number of controls, plus an additional 1,000 people from affected families. Mayeux said that using this data, his group has found six loci that may explain why the age at onset ranges so widely, from 45 to 75 years, in people who carry the G206A presenilin 1 mutation.
Other researchers have begun combining genetics with other measures to hunt risk alleles. In a short talk, Philip De Jager, Brigham and Women's Hospital, Boston, outlined an epigenomics approach to identifying risk factors for AD. De Jager, together with David Bennett at Rush University, Chicago, has correlated DNA methylation and micro RNA expression in the dorsolateral prefrontal cortex with the presence of amyloid plaques in the brain. They determined DNA modification, postmortem, at more than 400,000 CpG-rich, methylation-susceptible sequences in people from the Religious Orders Study and the Memory and Aging Project being conducted at Rush. Of 740 participants, almost half had been diagnosed with AD, a quarter had mild cognitive impairment, and the others were cognitively normal when they died. In people with plaque pathology, 117 regions were differentially methylated and 13 nearby genes were differentially expressed, De Jaeger said. A replication study using samples from a Mayo Clinic cohort confirmed the association for seven of those genes: Bin 1, ACACB, AP3M2, DDB1, DDOST, DLL1, and Slc17a7. A Bin1 single-nucleotide polymorphism (SNP) had previously come up in GWAS studies, but De Jager said that was unrelated to methylation changes. Interestingly, these methylation and expression changes occurred in asymptomatic individuals, suggesting an early event in disease.
Using a similar approach, De Jager correlated plaque pathology with micro RNA expression. Postmortem tissue samples from 711 people revealed that 25 of 309 miRNAs tested were expressed differently in people with amyloid pathology than in those without. Analysis of target genes for those miRNAs suggests that they may be up- or downregulated, as well. Combining this with the methylation data, the researchers are building a network of methylation sites and miRNAs that drive expression changes associated with pathology. De Jaeger said that miR-1260 emerged as a master regulator. Keystone attendees considered this approach attractive for other pathologies, such as Lewy bodies or TDP-43 inclusions.
What about known risk variants? Scientists at the meeting agreed that they need to better understand how those lead to disease. Investigators presented clues obtained by weaving in data from protein interaction networks and expression analysis. “Genetics doesn’t need to happen in a vacuum anymore,” said Singleton. “Integrating data is key to proving association and understanding function.”
In some cases, the integration starts with digging deeper into the data already available. “GWAS filter out all but the most robust hits,” Gasser told Alzforum, explaining that among the genes that don’t make the grade could lie true associations that act within the guise of a larger network. Sifting through 249 near-misses from the GWAS conducted by the International Parkinson’s Disease Genomics Consortium (IPDGC), Gasser found that half of them were related to the immune system. This allowed his team to generate an “inflammatory subtype” based on 100 SNPs and use that to stratify PD patients. The scientists subsequently screened the blood of 300 PD patients from the GWAS. They found higher levels of the key inflammatory cytokine IL-6 in patients harboring the inflammatory genetic fingerprint than in those who did not, indicating that the genetic association translated into a phenotype. While the findings are preliminary, Gasser said such experiments have the potential to group patients into different disease categories that could lead to more personalized treatment. “We’re not going to look at PD patients as one big pool anymore,” Gasser told Alzforum. His lab is currently stratifying patients based on a genetic subtype of mitochondrial dysfunction, as well.
Besides tapping networks to link GWAS hits to functional changes, researchers can learn from focusing on individual hits. For example, GTP cyclohydrolase 1 (GCH1), a gene involved in the production of dopamine and linked to dopamine-responsive dystonia (DRD), popped up as a risk factor in the IPDGC GWAS. As with a majority of those hits, the GCH1 variant fell into a non-coding region. However, comparing exome-sequencing data from PD patients and controls, researchers led by Niccolo Mencacci at University College London unearthed 10 rare variants within GCH1’s coding region. The new finding strengthens the idea that defects in dopamine production are a cause of PD, rather than just a symptom, in some people, Gasser said.
With GWAS hits in non-coding regions and rare variants in the coding region, the genetic architecture of GCH1 represents the norm for PD risk factors, Gasser said. Variants in non-coding regions likely control expression levels of nearby genes, but how they modulate expression or even which genes they affect can’t be determined by GWAS data alone.
“There are probably 20 to 30 loci from PD GWAS studies, each one lying near 20 to 30 genes,” commented Mark Cookson of the NIA. “So we have to ask: What’s our best shot?” Answering that question will require smart priorities, Cookson said. Schellenberg agreed this will be challenging. Of the AD GWAS hits to date, 90 percent are in non-coding regions, and it will be difficult to predict which genes they control. “We know that only 20 percent of regulatory elements affect the closest gene,” he said. On top of that, while the mean distance between regulatory element and target gene is about 120 kilobases, it can be 1.4 megabases or farther, said Schellenberg. Layering on more complexity, he reminded the audience that multiple enhancers can affect a given gene and a single enhancer can affect multiple genes, not always to the same extent. "The challenge of translating GWAS data is to identify which gene is relevant and determine whether risk variants increase or decrease expression," he said.
Taking a stab at this, Vincent Plagnol of University College London studied changes in gene expression associated with 16 established PD risk loci identified by GWAS. Using microarray analysis of brain samples from healthy controls, Plagnol searched for expression changes of genes thought to be under control of the GWAS SNPs. Of the 16 non-coding hits, only three correlated with nearby expression quantitative trait loci (eQTLs), which are genomic regions that regulate gene activity. In other words, three of 16 GWAS hits —RAB7L1, SPPL2B, and GPNMB—affected expression of nearby genes. However, Plagnol cautioned that his study was underpowered to find eQTLs that may affect expression of genes farther away. Larger sample sizes and RNA sequencing methods, rather than eQTL screening, would be necessary to uncover such variants (see Sept 2013 news story).
EQTL studies should serve as a prerequisite for moving forward with more costly functional studies, Plagnol said. “We shouldn’t base functional studies on GWAS hits alone,” he told Alzforum. Plagnol urged labs to readily share eQTL data.
This kind of work is crucial for understanding genome-wide associations, said Patrick Lewis of the University of Reading in England, who co-organized the Parkinson's disease meeting. It adds weight to various GWAS signals, and moves us closer to figuring out what they’re doing at the cellular level, he said.
The non-coding GWAS hits associated with LRRK2 and SNCA did not turn up in Plagnol’s eQTL screen. This is disappointing because those two genes are also known for rare Mendelian coding variants that cause PD. However, in a recent study, Plagnol did identify two exons in LRRK2 that appeared to be upregulated in people carrying a nearby non-coding variant not associated with PD (see Trabzuni et al., 2013). Why only two of LRRK2’s 51 exons were upregulated by the variation remains to be seen, Plagnol said.
“We are struggling to understand the genome-wide association at the LRRK2 locus,” said Lewis, who was a co-author on Plagnol’s LRRK2 mapping study. “It doesn’t seem to be an eQTL. It may be a splicing locus, but at the moment that really isn’t clear.”
Cookson commented that because the brain expresses low levels of LRRK2, correlating it to eQTLs could prove difficult. However, he noted that the expression analysis is a mathematically rigorous way to prioritize variants, and could bring researchers closer to understanding which ones play a role in disease.
For his part, Cookson took a different approach to clarify LRRK2’s murky role in PD. He presented recently published data from an unbiased screen of LRRK2 protein-interaction partners. It pinpointed Rab7L1, a previous GWAS hit and a gene Plagnol had tagged as an eQTL (see Feb 2014 news story). Cookson identified other proteins that interact with LRRK2 and Rab7L1: GAK, Hsc70, and BAG5. All play a role in the clearance of vesicles in the trans-Golgi network. The lab is now seeding protein arrays with LRRK2’s interaction partners to expand the network. The synergy between GWAS, eQTL, and protein-interaction studies in identifying Rab7L1 gave Cookson hope for progress. “Rab7L1 is a pretty good candidate in that locus. When we combine these multiple prioritization strategies, something will rise to the top,” Cookson said.—Jessica Shugart and Tom Fagan.
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