Taos: Biomarkers Keep Blooming, But Where’s the Fruit?
Biomarkers could be invaluable diagnostic and prognostic indicators for Alzheimer’s and other neurodegenerative diseases. Hardly a meeting goes by these days without mention of a new potential marker. Neurodegenerative Diseases: The Molecular and Cellular Basis of Neurodegeneration, a Keystone Symposium held 21-26 February 2011 in Taos, New Mexico, was no exception. Plasma, cerebrospinal fluid, imaging, and antibody markers were all being discussed. But lest anyone get too optimistic, there also were sobering thoughts about markers in general, not least being a reminder of how few have actually been approved for diagnostic use.
John Trojanowski, University of Pennsylvania, reviewed the biomarker data that has emerged from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). There is consensus among AD researchers that ADNI has been a success, and most researchers were happy to see that funding was found to continue the project with ADNI2 (see ARF related news story). One thing to have emerged from ADNI is the potential for finding markers for various stages of AD, from pre-symptomatic to mild cognitive impairment to AD itself, said Trojanowski. He stressed that big science projects such as ADNI, and more recently, the Parkinson’s Progression Markers Initiative (see ARF related news story) are crucial to moving the field forward, not least because they can address technical issues such as assay standardization and validation. Simply finding a potential marker is not enough, he stressed. A recent Nature review on the subject by George Poste, Arizona State University, Scottsdale, surveyed the literature to find some 150,000 papers documenting thousands of biomarkers, of which fewer than 100 are used in routine clinical practice (Poste, 2011). Trojanowski said that many of these biomarkers never make it to the clinic because methods are not standardized, which makes validation difficult. Leroy Hood, president of the Institute for Systems Biology, Seattle, Washington, echoed this view. While Hood sees great promise for medicine in the twenty-first century, including the ability to cut medical care costs, he said that the fundamental challenge for medicine is to deal with incredible complexity. When researchers start to deal with large datasets, noise, both biological and technological, becomes a huge concern, he said. He predicted, for example, that many of the hits that have come from large genomewide association studies will turn out to be noise.
David Holtzman, Washington University, St. Louis, Missouri, also emphasized the methodological challenges in searching for meaningful biomarkers. His lab has been conducting high-throughput, unbiased proteomic screens of cerebrospinal fluid (CSF) and plasma. In Taos, he stressed that reproducibility of CSF analysis can be problematic, and that replication of the analysis is essential. He described new potential markers that came from a CSF proteomic screen. One of them, YKL-40, has no known function but might be a marker of inflammation. Produced by astrocytes, it strongly correlates with tau in CSF, said Holtzman, and his group has validated the marker in hundreds of samples by ELISA. When combined with Aβ42, it predicts who will progress from normal to cognitively impaired over five years as well as does the tau/Aβ42 ratio (see Craig-Schapiro et al., 2010). A second marker, visinin-like protein-1 (VILIP-1), does even better. It, too, is elevated in the CSF of people with AD compared to normal controls or people with non-AD dementia. It better predicts conversion to cognitively impaired when combined with Aβ42 than does tau in their dataset. Holtzman said this was striking over a four-year period. VILIP-1, a cytosolic calcium-binding protein, is found in neurons. VILIP-1 CSF levels also correlate with those of tau. This led Holtzman to propose that tau is elevated in AD CSF, not because of the presence of neurofibrillary tangles that comprise tau, but because synaptic or neuritic degeneration releases proteins like tau and VILIP-1 into the parenchyma.
In response to a question from the audience about whether YKL-40 is a marker of general astrogliosis, Holtzman said that his lab looked at CSF samples taken from about 30 people with other neurodegenerative disorders, including frontotemporal dementia and progressive supranuclear palsy. In most samples, neither YKL-40 nor VILIP-1 was elevated. This could be because those disorders involve fewer neuritic changes than does AD, he suggested. Others suggested that not all glioses are the same, and that markers of the process may not necessarily be common among different disorders.
Several poster presentations from Michael Sierks’s group at Arizona State University in Tempe described a different type of CSF analysis. Sierks uses a combination of phage display and atomic force microscopy to generate and identify antibody fragments (nanobodies) that react with different oligomeric proteins. The field is still grappling with ways of identifying such oligomers in biological samples, and some of them could turn out to be early diagnostic markers. Sierks isolated several nanobodies to oligomers of amyloid-β (including A4) and α-synuclein (including D5 and 10H). The D5 nanobody bound to antigens in CSF of PD patients and AD patients, but not of normal controls or of people with multiple system atrophy, a synucleinopathy that is difficult to diagnose. The A4 nanobody recognized antigens specifically from the CSF of AD patients. Though the sample sizes were small, the technology may prove useful for identifying specific oligomeric species in human tissue. Eliezer Masliah told ARF that the technology is promising; Masliah is collaborating with Sierks to study α-synuclein oligomers. On a separate poster, Sharareh Emadi in Sierks’s lab showed that D5 and another nanobody, 10H, react with distinct forms of α-synuclein and also with different cell types. The D5 nanobody reacts with smaller antigens and with dopaminergic cells, while 10H reacted with larger oligomers and with both dopaminergic and cholinergic cells. The differential reactivity of the nanobodies suggests that specific forms of oligomeric α-synuclein may form in different cell types.
Moving from molecules to whole-brain imaging, Erik Musiek from the University of Pennsylvania, Philadelphia, described a comparison between fluorodeoxyglucose (FDG) PET and arterial spin labeling MRI as measures of brain activity. FDG-PET is a good tracker, said Musiek, but it is expensive and the radiation limits repeat measurement. Arterial spin labeling MRI is not invasive, is cheaper, and can be carried out repeatedly with little risk. Whereas FDG-PET measures brain activity based on glucose consumption, arterial spin labeling (ASL) does it by measuring blood perfusion. While data indicate ASL-MRI is altered in AD patients, no one had ever before done a head-to-head comparison with FDG-PET, said Musiek.
He described a small pilot study that did just that. Eighteen control volunteers and 16 AD patients were injected with FDG while undergoing ASL scans, and were then immediately taken for PET scans of glucose consumption. Musiek found that both techniques lit up comparable areas in the brains of controls. In AD brains, regions where FDG-PET showed deficits matched with those showing perfusion deficits by ASL. Quantitatively, AD patients had a 31 percent decrease in ASL compared to controls; that difference was 14 percent with FDG-PET. Voxel-based analysis showed good matching between areas that showed reductions in both types of analysis. Musiek concluded that this small pilot study suggests that ASL and FDG-PET perform with similar diagnostic accuracy. He said ADNI2 volunteers will undergo ASL, and analysis from them should help confirm if the technique has clinical potential.—Tom Fagan.
- Research Brief: Announcing ADNI2—Funding for Five More Years
- PPMI: Parkinson's Field’s Answer to ADNI
- Poste G. Bring on the biomarkers. Nature. 2011 Jan 13;469(7329):156-7. PubMed.
- Craig-Schapiro R, Perrin RJ, Roe CM, Xiong C, Carter D, Cairns NJ, Mintun MA, Peskind ER, Li G, Galasko DR, Clark CM, Quinn JF, D'Angelo G, Malone JP, Townsend RR, Morris JC, Fagan AM, Holtzman DM. YKL-40: a novel prognostic fluid biomarker for preclinical Alzheimer's disease. Biol Psychiatry. 2010 Nov 15;68(10):903-12. PubMed.
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Taos: Redox Redux—Putting the Oxidative Stress Theory to the Test
Suggesting that oxidative stress precipitates cell death in neurodegenerative disease is one thing; proving it is quite another. If seeing is believing, then researchers may have brought us one step closer. At Neurodegenerative Diseases: The Molecular and Cellular Basis of Neurodegeneration, a Keystone Symposium held 21-26 February 2011 in Taos, New Mexico, Brian Bacskai of Massachusetts General Hospital, Charlestown, showed that cell death does indeed follow oxidative stress in normal mice, but more so in models of Alzheimer’s disease. Bacskai presented work performed by postdoctoral fellow Hong Xie using a redox-sensitive reporter to illuminate oxidative stress in the brains of living mice.
Bacskai and Xie used a modified green fluorescent protein as a sensor of cellular oxidation potential. The protein, called roGFP, developed by Jim Remington at the University of Oregon, contains two cysteine residues that form a disulfide bond under oxidative conditions. When the bond forms, the fluorescent properties of the protein change, such that it and the reduced GFP glow best when excited by light of different wavelengths. Bacskai and Xie targeted roGFP constructs to neurons in normal mice and in a double-transgenic mouse model of AD (APPSw/PS1ΔE9), which begins to form amyloid plaques at about four to five months of age. They aimed a two-photon microscope through a transcranial window, in essence visualizing cellular oxidation in the living brain.
The microscopy revealed both good news and bad news. The good news is that very few cells in normal mice are sufficiently stressed to activate the reporter. Of some 50,000 neurons analyzed, only 0.13 percent glowed green from oxidized roGFP. Analysis of the APP/PS mice was promising as well, in that the total number of oxidized cells was still relatively small (about 0.42 percent). The bad news was what happens to neurons in the vicinity of amyloid plaques. Bacskai showed that a halo of oxidation surrounds plaques, where almost 60 percent of neurites glowed green. Over a two- to three-month period, the oxidative stress only got worse, even in the absence of plaque growth.
What happens to an oxidized cell? To answer this, Bacskai and Xie imaged individual neurons longitudinally. Over two months, a substantial number of oxidized cells disappeared in the vicinity of plaques. Curious to know how quickly neurons succumb, they looked more frequently and were surprised to see that some cells that became oxidized vanished within a few hours. In fact, Bacskai said that once a neuron becomes oxidized, it is doomed to die, and does so within 24 hours. After his talk, he told ARF that he is not sure if new neurons can replace those lost. This could be important in both AD and in normal mice. Even with oxidation rates as low as 0.13 percent, it would not take long before there was massive neuron loss in the normal mouse brain if oxidation precipitated cell death within a day.
Bacskai said he was confident that the oxidized cells were, in fact, dying and not just losing their reporter or modulating their redox potential. Co-staining for nuclei and caspase activity showed that oxidized cells underwent apoptosis, or programmed cell death, losing their nuclear material into the bargain. In fact, stopping the cell death program was difficult. Molecules that trap free radicals and reduce oxidative stress failed to prevent oxidation when given daily for a month. Antibodies to amyloid-β fell short, too, though these were only applied for an hour. Perhaps a longer treatment might have some effect, Bacskai said. One thing that did work well when applied directly to the brain was dithiothreitol (DTT), a reducing agent that breaks disulphide bonds.
This talk generated many interesting questions. Some noted that the APP/PS model shows little spontaneous neuronal loss, and wondered if this was the best model of oxidative stress and cell death for AD. Bacskai agreed that another model could prove interesting, but stressed that this was simply to test what happens during amyloidosis. Others questioned the use of the roGFP reporter itself. It seems to respond only to extremely high oxidation potentials (another reporter might detect milder oxidative stress), and once oxidized it is not clear if it can be reduced in vivo by naturally occurring reducing agents. Some wondered whether oxidative stress and death were related to the depth of a cell in the cortex. Since the two-photon microscopy does not penetrate deeply, Bacskai has only looked at cortical layers I and II; he has not looked for correlations between depth and apoptosis.
Jim Surmeier, Northwestern University, Chicago, Illinois, asked about the relationship between calcium and oxidative stress. Bacskai said it would be interesting to look into this, particularly which comes first. Scientists have long suspected that calcium poisons neurons in AD (see ARF related news story). Surmeier reported some years back that a particular type of voltage-gated calcium channel could explain why dopaminergic neurons in the substantia nigra are particularly susceptible in PD (see ARF related news story). At Taos, Surmeier outlined results published late last year in Nature based on targeting the roGFP reporter to the mitochondrial matrix of dopaminergic neurons. He found that calcium entry through L-type calcium channels during normal pacemaking increased mitochondrial oxidant stress in vulnerable dopaminergic substantia nigra neurons, and that loss of DJ-1 exacerbated this oxidant stress (see ARF related news story). DJ-1 loss-of-function mutations are associated with early onset Parkinson’s disease.—Tom Fagan.
- Follow the Calcium—Influx Through NMDARs Releases Yet More From ER
- Teaching Old Neurons Young Tricks—“Rejuvenation” Protects from PD
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Taos: Not the Usual Suspects: PARIS et al. Mark Progress in PD
In the movie Casablanca, protagonists Rick and Ilsa would always have Paris. Parkinson’s disease researchers always will as well, if a different one. Parkin interacting substrate (PARIS) was not born of the movie screen, but of a screen for parkin substrates. Its debut was a highlight at Neurodegenerative Diseases: The Molecular and Cellular Basis of Neurodegeneration, a Keystone Symposium held 21-26 February 2011 in Taos, New Mexico. PARIS may be a lead actor in mitochondrial dynamics, an underlying theme at the meeting. In particular, it seems that researchers are finally getting a grip on the long-suspected link between mitochondria and not only PD, but other neurodegenerative diseases as well. Many molecular players take part in this link, and quite a few appear to have a penchant for regulating fission and/or fusion of these organelles. This story features new data on a large cast of characters involved. It opens with PARIS and parkin, then moves on to PGC1α and Pink1, mitochondrial anchored protein ligase (MAPL) and Drp1, then further on to mitofusin, PARL, ataxin-3, and Vms1. These proteins each play a part—some large, some small—in the molecular underpinning of mitochondrial deficits in PD and related conditions. So sit back and let this molecular biology revue pass before your eyes as Alzforum closes its Keystone coverage.
First, consider parkin. Though this gene was one of the first to be linked to Parkinson’s, what the protein does in the disease has remained a bit of a puzzle. It is a ubiquitin ligase with so many substrates that it is uncertain which is most important to pathology, said Ted Dawson, Johns Hopkins University, Baltimore, Maryland. Now, there is good reason to think that PARIS might be one of the important ones, he suggested. Originally isolated by yeast two-hybrid analysis using parkin as bait, PARIS co-immunoprecipitates with parkin, co-localizes with it in cortical neurons, and binds to the parkin RING domains that are crucial motifs in many ubiquitin ligases. Parkin ubiquitinates PARIS in neuroblastoma cells, and familial parkin mutations that cause PD block this modification, said Dawson. Furthermore, his group rescued PARIS ubiquitination deficits in parkin knockdown cells by overexpressing wild-type parkin in the cells.
Merely being a parkin substrate does not mean a protein plays a role in PD pathology, but Dawson reported high levels of PARIS in brain tissue from both familial and sporadic PD. In the former, the scientists saw this in the cingulate cortex, but did not have enough material to test brain regions most susceptible to pathology, such as the striatum and substantia nigra. In sporadic cases, PARIS was up twofold in both of these locations, but was normal in the cerebellum and frontal cortex, areas that stay relatively unscathed long into this disease. The pattern of change suggests a link between PARIS and PD at the protein level, Dawson said, as PARIS messenger RNA was unchanged.
PARIS sports zinc finger domains as well as a Kruppel-associated box, and proteins containing both are usually transcriptional repressors, said Dawson. It turns out PARIS binds to a specific form of insulin response sequence. One gene that shares this sequence is PGC1α, a mitochondrial protein that may protect against Parkinson’s (see ARF related news story) and even Alzheimer’s (see ARF related news story). In Taos, Dawson showed that PARIS repressed expression of PGC-1α, and that parkin rescued this suppression. In parkin knockouts, on the other hand, PARIS levels rise and PGC-1α drops, leading to dopaminergic cell death, which is prevented by knockdown of PARIS. These findings suggest that excess PARIS contributes to the demise of dopaminergic neurons when parkin is mutated, suggested Dawson. This work appeared in the March 4 issue of Cell (Shin et al., 2011).
You Say Fission—I Say Fusion
Whether PARIS links parkin to mitochondrial defects is not yet clear, but perturbation of mitochondrial morphology has been a topic of great interest among Parkinson’s researchers. Both parkin and the product of another PD gene, the kinase Pink1, somehow toggle the balance between mitochondrial fission and fusion (see ARF related news story and ARF news story). To work out the mechanism, Ming Guo, University of California, Los Angeles, uses fruit fly muscle as a model system. Muscle cells are highly dependent on mitochondria, and in Taos, Guo showed that they have a similar phenotype to neurons when Pink1 or parkin are mutated or absent.
Guo described genetic screens to identify components of the fission/fusion pathways that might interact with parkin and Pink1. From this exercise emerged the mitochondrial anchored protein ligase (MAPL). Recently discovered elsewhere (see Braschi et al., 2009), MAPL is one of the family of ligases that tag other proteins with small ubiquitin modifiers (SUMOs). Guo reported that MAPL seems to work similarly to the protein Drp1, in that it promotes fission and attenuates Pink1 mutant phenotypes in both muscles and dopaminergic neurons. MAPL seems to work through Drp1, said Guo, and there is evidence that the ligase SUMOylates Drp1. MAPL also rescues Pink1 mutants in the absence of SUMOs, however, so SUMOylation may not be the full story, Guo said.
On the other side of the coin—that is, fusion—evidence is emerging that parkin/Pink1 perturbs that as well. The mechanism involves ubiquitinating mitofusin, which, as the name suggests, promotes mitochondrial fusion. Scientists in Richard Youle’s lab at the National Institute for Neurological Disorders and Stroke, Bethesda, Maryland, reported last year that small compounds that uncouple mitochondrial electron transport from ATP synthesis cause a buildup of Pink1 in mitochondria. The theory is that uncoupling reduces the voltage potential across mitochondria that fuels transport of Pink1 from the outer to the inner mitochondrial membrane, where normally it is quickly degraded. Stuck on the outer membrane, Pink1 then recruits parkin. In Taos, Youle added some of the missing pieces to the puzzle, namely, the protease that normally degrades Pink1 and a potential substrate for parkin. The former is PARL, or presenilin-associated rhomboid-like protein. When the researchers take PARL out of the picture, they prevent the normally constitutive degradation of the Pink 1 kinase (see Jin et al., 2010). The parkin substrate is mitofusin. When mitochondria are uncoupled, extracts contain mitofusin protein of progressively larger sizes, indicative of ubiquitination.
As these studies show, researchers are beginning to unravel manifold links among parkin/Pink1, mitochondrial dynamics, and Parkinson’s pathology. The findings could have broader ramifications, since mitochondrial damage is implicated in other neurodegenerative diseases. For example, Machado-Joseph disease (MJD), one of the most common forms of ataxia, is caused by a polyglutamine (PolyQ) expansion in ataxin-3. In Taos, Thomas Durcan, who works in Edward Fon’s lab at McGill University, Montreal, Canada, noted that many people with MJD have symptoms of Parkinson’s. Could this be because ataxin-3, a de-ubiquitinating enzyme, interferes with parkin, the ubiquitin ligase? That is exactly what Durcan and colleagues reported earlier this year. Ataxin-3 clips ubiquitin off parkin and promotes its clearance from cells via autophagy. In effect, ataxin-3 could be acting as a parkin loss-of-function mutation, Durcan said. Interestingly, it is not the wild-type form of ataxin-3 that de-ubiquitinates parkin, but PolyQ expanded forms (see Durcan et al., 2011). In Taos, Durcan added another twist. PolyQ-ataxin-3 increases oxygen consumption in cells without boosting ATP synthesis. This is just what mitochondrial uncouplers do, bringing to mind Youle’s work. PolyQ ataxin might be trapping Pink1 on the mitochondria and recruiting parkin, which then drives mitochondrial autophagy (see ARF related news story).
One of the challenges of studying mitochondria in disease, noted Jin-Mi Heo, is that a fourth of mitochondrial proteins are characterized either poorly or not at all. Working in Jared Rutter’s lab in the University of Utah School of Medicine, Salt Lake City, Heo reported on one protein that might be part of a novel mitochondrial degradation pathway. Vms1, which stands for VCP/Cdc48-associated mitochondrial stress responsive 1, partners Cdc48 and Np14, components of the ubiquitin-proteasome system. The two are known to help clear proteins associated with the endoplasmic reticulum. Do they help dispatch mitochondrial proteins as well? Heo found that when mitochondria are stressed, Vms1 recruits Cdc48/Np14 to the organelles. When Vms1 is absent, ubiquitinated proteins accumulate in mitochondria, the organelles lose function, and the cell soon dies. It seems that Vms1 is necessary for maintaining a healthy mitochondrial pool. Whether it may be important for Pink1/parkin-induced mitophagy, or figure in any neurodegenerative disease, remains to be seen.
As this parade of presentations revealed, a growing list of proteins is beginning to fill the nexus between neurodegeneration and mitochondria. Most of them drive (e.g., Pink1, parkin, mitofusin, Drp1, MAPL, polyQ-ataxin-3) or are driven by (e.g., PARIS, PGC-1α) fission- or fusion-related events. At the end of Casablanca, of course, a police roundup of the usual suspects failed to nab the culprit. Have researchers done better, or are the major players yet to be discovered?—Tom Fagan.
- Gene Captain, PGC-1α, Abandons Mitochondria in PD
- Special Issue Explores Link Between Metabolic Disease and Dementia
- Pink Fission—Serving Up a Rationale for Parkinson Disease?
- Abnormal Mitochondrial Dynamics—Early Event in AD, PD?
- Shin JH, Ko HS, Kang H, Lee Y, Lee YI, Pletinkova O, Troconso JC, Dawson VL, Dawson TM. PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson's disease. Cell. 2011 Mar 4;144(5):689-702. PubMed.
- Braschi E, Zunino R, McBride HM. MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission. EMBO Rep. 2009 Jul;10(7):748-54. PubMed.
- Jin SM, Lazarou M, Wang C, Kane LA, Narendra DP, Youle RJ. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J Cell Biol. 2010 Nov 29;191(5):933-42. PubMed.
- Durcan TM, Kontogiannea M, Thorarinsdottir T, Fallon L, Williams AJ, Djarmati A, Fantaneanu T, Paulson HL, Fon EA. The Machado-Joseph disease-associated mutant form of ataxin-3 regulates parkin ubiquitination and stability. Hum Mol Genet. 2011 Jan 1;20(1):141-54. PubMed.