Sorrento: Immunotherapy Update Hot Off Lectern of AD/PD Conference
Yesterday, the 7th International Conference on Alzheimer’s and Parkinson’s disease—AD/PD 2005 for short—drew to a close in beautiful Sorrento, Italy. Nearly 1,500 scientists from 52 countries met to brush up on news and trends in research and treatment for neurodegenerative diseases. Abraham Fisher from the Israel Institute for Biological Research in Ness Ziona, and Israel Hanin at Loyola University, Chicago, have organized this biannual conference for the past 20 years. They include co-organizers from a different host country each time, this year they were Maurizio Memo from University of Breschia, and Fabrizio Stocchi from La Sapienza University in Rome. AD/PD 2005 took place in a hotel built into a Sorrento mountainside amid lemon and orange orchards. The setting offered sweeping views over an ancient town that is built spectacularly into the rocks of a peninsula rounding off the southern tip of the gulf of Naples.
The Alzforum news team will post news reports of this conference for the next two weeks. This first installment of our hot-off-the-podium dispatches summarizes the status of AD immunotherapy research. The setback in the AN 1792 trial cast a temporary pall over the field (see Alzforum Live Discussion), but soon after, researchers began trying again and the approach remains one of the most closely watched in the field. The Alzforum has covered this science in detail (for background, see Can a Shrinking Brain Be Good for You?, Vaccine, Microglia, NGF News Fill in Neuroimmunology Picture, Immunotherapy—The Game Is Still in Town); therefore, this story focuses on new human data and experimental approaches.
Dale Schenk of Elan led off an immunotherapy symposium by noting second-generation approaches his company is currently exploring. They include passive vaccination—Elan is completing a phase 1 trial of a humanized monoclonal antibody to Aβ and is preparing a phase 2 at several sites in the U.S.—and preclinical work on active immunogens made by conjugating small Aβ fragments to an external carrier. In parallel, investigators in many other laboratories are developing their own approaches.
An area that requires extensive investigation concerns the mechanisms that drive the distribution of Aβ between its different pools in plasma, CSF, and the brain. Adding antibodies to this already complicated process creates a level of complexity that research has not yet fully understood, Schenk said. For example, in the AN1792 trial, changes in Aβ levels followed no consistent pattern, which may be due to different antibodies being produced. Another major question in AD immunotherapy revolves around how amyloid gets cleared; different groups have proposed different routes including microglia devouring amyloid and the peripheral sink hypothesis. “This may be one of those questions where everyone is right,” Schenk said.
Those who perched on the edge of their seat expecting new clinical data eased back in disappointment when Schenk did not present any. He merely noted that further analysis of the phase 2 patients showed that the patients who had the strongest antibody response also tended to perform a tad better on some of the functional assessments, and that the tau levels in their cerebrospinal fluid had come down the farthest. This adds preliminary human data to an emerging body of animal data suggesting that tau pathogenesis occurs downstream of amyloid pathology and that amyloid removal can hold its early stages at bay. It is also the first demonstration of any therapeutic intervention that can lower tau in people with AD, Schenk noted.
Schenk added that, looking back, mice have had a mixed record of predicting what would happen in humans. The mouse data predicted plaque removal correctly. Their prediction of functional improvement remains largely untested, though immunotherapy buffs are putting hope into preliminary hints at a slight functional improvement in a few AN-1792 participants. On safety, the mouse models failed to predict the meningoencephalitis side effect while, on the other hand, they exhibit a bleeding side effect in the brain that has, to date, not been detected in any of the treated people (see Racke et al., 2005; Pfeifer et al., 2002; scroll down for Morgan’s presentation).
Nick Fox, of London’s University College, who had presented the MRI imaging results of the AN-1792 cohort last July in Philadelphia (see conference news story,) noted that they are slated to appear in the journal Neurology in May. He repeated that analyzing the surprising shrinkage in brain volume against other measures in the trial suggests that people whose brains had shrunk the most were also the ones who had the greatest drop in CSF tau levels. They also performed a tad better on some of the functional measures. The next challenge, besides avoiding encephalitis in future trials, is to understand what underlying processes in the brain reduce its volume after immunization, as amyloid removal alone can account at best for a fraction of the observed shrinkage. Fox suggested that fluid shifts may be part of the answer, while Roger Nitsch of University of Zurich suggested in his talk that the calming down of astrogliosis might free up measurable space.
Nitsch’s group perform clinical MRI on the Swiss cohort themselves. Christoph Hock, a longtime colleague of Nitsch’s, said that data from the year three imaging time point are not available yet. The year three clinical follow-up is being completed. Hock noted that some patients still have high antibody titers at this point, and that these “responders” still outperform the “non-responders” slightly on functional assessments. Addressing the question of which Aβ measurements are best suited to such trials, Hock said that the Zurich researchers observed patients with high ELISA titers decline but not those with high TAPIR values, suggesting that using ELISA alone might scramble the trial result and understate how well the responders did. (The TAPIR is an assay developed in Roger Nitsch’s group, where patient serum is used to stain AD tissue. Its developers claim that it is better at detecting pathogenic neoepitopes than standard ELISA tests.) Some researchers had deplored that the whole AN-1792 trial population was not being followed properly, but Hock said that TAPIR analysis of the whole cohort is under way.
James Nicoll presented his analysis of a fourth postmortem case in addition to the three that have to date been published (Nicoll et al., 2003; Ferrer et al., 2004; Masliah et al. 2005). Case 4 was a British patient from the phase 1 trial of the AN1792 vaccine, who died four months after receiving it of an abdominal aortic aneurysm deemed unrelated to immunization. Unlike the other three cases, whose cortex was devoid of plaques in some areas and whose Aβ burden had dropped, case 4 had still had abundant amyloid plaques and the amyloid load was like that of unimmunized patients. However, the plaques looked different, Nicoll said. They appeared looser, moth-eaten, and nearby microglia carried lots of Aβ inside their lysosomes. Overall, it looked as if the patient had died during an active phase of plaque removal, Nicoll said.
Nicoll also talked about tau pathology, and noted that in cases 1, 2, and 3, tangles and neuropil threads stayed in place but dystrophic neurites had straightened out, as was reported in mice, as well (Brendza et al., 2005). This was yet another of many hints reverberating throughout the meeting that amyloid immunotherapy may resolve early stages in tau’s pathogenic pathway. (Other work in mice, most prominently some presented by Mike Hutton of the Mayo Clinic in Jacksonville, Florida, goes in the same direction. Hutton reported that shutting off a tau transgene in an inducible mouse model enabled the mice to recover from their memory deficit even while tangles stayed in place but dystrophic neurites resolved.)
Finally, Nicoll addressed the current worry that Aβ immunotherapy may remove parenchymal Aβ but cause trouble at the brain’s blood vessels. As a group, cases 1 through 4 had fairly severe cerebral amyloid angiopathy compared to what is typically seen in AD, Nicoll said. He is currently studying three additional autopsy cases from the phase 1 trial and gathering informed consent from the UK participants in these studies. “I want to reassure you that we are following up the UK patients,” he said.
Dave Morgan’s current work with mice ties into this concern about CAA, and its possible consequence of bleeding from damaged blood vessels. Morgan, who is at University of South Florida in Tampa, said that when his coworkers injected an anti-Aβ antibody into APP Tg2576 mice, this passive vaccine not only elicited a strong antibody response but also led to an increase in CAA. “This surprised us,” Morgan said. Further study of the different types of deposit in the mice showed that, after both three and five months of vaccination, parenchymal amyloid predictably decreased but vascular amyloid increased to where it became the predominant deposit, where previously most was parenchymal. Morgan speculated that microglia absorb amyloid in the parenchyma and dump it out at the blood vessels, where its amounts overwhelm local transport and drainage mechanisms so that it promptly deposits again. Dosing the vaccine to a slower rate of amyloid removal might address this problem, he said (see also Weller talk in St. Moritz meeting report).
Intrigued by Mathias Jucker’s research of micro-hemorrhages (Pfeifer et al., 2002) and Dave Holtzman’s work on ApoE and CAA (Fryer et al., 2003), Morgan decided to look for leaking blood vessels in the immunized mice with the dye Prussian Blue. Indeed, he found that, over time, increasing amounts of blood seeped out of blood vessels into the brain parenchyma. The mice seemed strangely unfazed by these micro-hemorrhages, as they continued to show a vaccine-induced improvement in the water maze test even as the bleeding increased. This may mean that the bleeds are harmless, or it might remind us how poorly water maze performance models human brain function. (Morgan used an antibody to the C-terminal end of Aβ; the current Elan passive vaccination trial uses an N-terminal antibody.)
Finally, Morgan raised the murky issue of what to make of microglial activation in AD. He noted that his most recent immunization studies provide more evidence that antibodies penetrate the brain, prompting microglia to express Fc antibody receptors. The time course of this transient increase parallels amyloid removal. Along with the Fc receptors, Morgan detected temporary changes in other microglial markers. Together, he said, this work paints a complicated picture of the microglial response over time, which belies a widespread assumption that microglial activation is always bad. Rather, Morgan believes he is observing a normalization of the pre-existing microligal response. In short, he suspects anti-Aβ antibodies may give errant microglia something useful to do.
One of the more promising new vaccination approaches lies in fashioning cleverer vaccines from small snippets of Aβ. That became necessary because scientists now agree that the side effect in the phase 2 trial arose from autoimmune Th1 lymphocytes. Egged on further by an adjuvant known to elicit Th-1 responses, these cells reacted to Aβ and infiltrated the brain. Broadly speaking, Th-1 immune responses tend toward cellular responses and predominate in autoimmune conditions, whereas Th2 responses drive humoral, i.e. antibody, immunity and tend to be seen in allergies. To an immunologist, the workup of the AN 1792 patients’ brains was simple, said Michael Agadjanyan of the Institute for Molecular Medicine in Huntington Beach, California: “The B cells are the good guys, the T cells are the bad guys.” To avoid rousing Th1 cells, several groups have buffed up their immunology skills and are now testing vaccines based on B cell epitopes. (Aβ houses B cell epitopes between its amino acids 1-15, and T cell epitopes in the middle and toward the C-terminal end.)
Working with David Cribbs at University of California, Irvine, Agadjanyan took a B cell epitope and fused it with a generic T cell epitope called PADRE, which serves to activate Th2 helper cells. They, in turn, help fully activate Aβ-specific B cells into antibody-producing cells, as a proper immune response is thought to require. The trick in this scheme is that the T cells are directed against PADRE, not Aβ, and that the B cells make antibody against Aβ, not PADRE. An added technical twist boosts the strength of the immune response by planting the vaccine on a "lysine tree". The performance of this vaccine in Balb/C is published (Agadjanyan et al., 2005). In Sorrento, Agadjanyan said that in the APP Tg2576 and triple transgenic AD mouse models, too, the vaccine generates a strong antibody response but no T cell response. The cytokine profile in the vaccinated mice was consistent with a Th2 response, not an autoimmune reaction, and the antibodies bound specifically to plaques from AD brain.
Cindy Lemere, of Brigham and Women’s Hospital in Boston, has developed different new vaccines that do not evoke an Aβ-specific T cell response. She uses the Aβ1-15 peptide, either multiple copies attached to a lysine tree (the "dendrimeric" vaccine) or two copies connected by a lysine spacer and attached to an RGD motif to boost immunogenicity. An added appeal of her approach lies in the fact that she delivers her vaccines through the nose, raising the distant prospect of nose drops against AD.
Lemere’s team first described the performance of these new vaccines in wild-type mice at last year’s Society for Neuroscience conference in San Diego. In Sorrento, Lemere added data from tests in APP-transgenic mice. Both vaccines elicited a humoral immune response that was much stronger than that generated by prior vaccines using full-length Aβ. The antibodies labeled plaques and vascular amyloid in a TAPIR assay in AD tissue. When immunizing Lennart Mucke’s J20 APP-transgenic mice with the new vaccines for 6 months, Lemere found that the plasma levels of total Aβ shot up midway through the study and then came down toward the end. Plaque deposition decreased strongly, and cerebral Aβ levels dropped, as well. The mice exhibited no side effects. Lemere said that she hopes to test the vaccine in triple transgenic mice to look for effects on tau pathology, and then test it in monkeys (see also Lemere et al., 2004.)
Only the mouth can rival the nose as the simplest entry point into the human body. Dangle the prospect of a little white pill, and drug companies will listen with more interest to an academic’s budding experimental therapy than if it requires injection. Enter Takeshi Tabira’s from the National Institute of Longevity Sciences in Obu, Japan. Tabira described his group’s development of an oral vaccine that exploits the intestinal immune system’s ability to generate responses that follow a Th2 pattern.
The immune system in the gut is often overlooked as the dull, puny cousin of the more formidable system in the spleen and lymph nodes. However, it is quite extensive, spreading through the 300 square meters of intestinal mucosa an adult carries coiled up in the belly. To avoid peptide digestion in the stomach, Tabira’s group fashioned gene therapy vaccines out of Aβ1-43 and Aβ1-21 packaged into adeno-associated virus (AAV). This virus is used in experimental gene therapy regimens but comes with its own safety concerns. (Various gene therapy approaches are in preclinical development, but in the clinic all gene therapy remains problematic.)
Tabira said that antibodies against the Aβ peptides appeared in the lamina propria of the upper part of the small intestine of young Tg2576 mice treated with the vaccine. The mice developed mostly IgG antibodies, and their serum stained plaques from AD tissue in a TAPIR assay. The vaccine inhibited amyloid aggregation in vitro and reduced Aβ deposition and burden in vivo. It also appeared to reduce levels of phospho-tau. T lymphocytes from these mice did not react to full-length Aβ; the mice showed no evidence of inflammation save for microglial markers, Tabira reported. Older mice receiving the vaccine produced fewer antibodies, but also exhibited a reduction of their amyloid burden and inflammatory markers such as TGF-beta1 and certain cytokines. Tabira noted that these effects followed a single oral dose and then persisted for weeks.
Anticipating safety concerns regarding the use of AAV, Tabira noted that the cells at the top of the intestinal mucosa turn over quickly. In mouse and rat, these dead cells exfoliate into the gut lumen and are excreted, whereas monkeys internalize the dead cells first and then degrade them. Tabira was unable to detect the AAV vector in other tissues of the mouse, but even so, this issue will require careful study before moving this approach into humans. The vaccine has entered testing in monkeys, Tabira noted.
Overall, the talks brought into focus these common themes and questions:
- Safer antigens seem to be effective in mice,
- questions around vascular amyloid and hemorrhages need attention,
- detecting reactivity in tissue may be a better way of quantifying the antibody response than ELISA,
- clearance mechanisms remain murky but microglia are part of it,
- amyloid immunotherapy might stem tau pathology,
- all eyes will be on the passive vaccine once it has entered phase 2.
The AD/PD meeting featured other talks on vaccination, such as an update by Beka Solomon on her approach using filamentous bacteriophages to deliver Aβ peptides through the nose (Solomon, 2005), but this writer was unable to attend them all. As always, meeting attendees are cordially invited to complete this update with their own notes and corrections.—Gabrielle Strobel.
- Philadelphia: Can a Shrinking Brain Be Good for You?
- St. Moritz: Part 5. Vaccine, Microglia, NGF News Fill in Neuroimmunology Picture
- New Orleans: Immunotherapy—The Game Is Still in Town
- St. Moritz: Part 3. This Research Isn't Folding Up: Genetics, Transport, Seeding, Protein Microscopy
- Racke MM, Boone LI, Hepburn DL, Parsadainian M, Bryan MT, Ness DK, Piroozi KS, Jordan WH, Brown DD, Hoffman WP, Holtzman DM, Bales KR, Gitter BD, May PC, Paul SM, Demattos RB. Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta. J Neurosci. 2005 Jan 19;25(3):629-36. PubMed.
- Pfeifer M, Boncristiano S, Bondolfi L, Stalder A, Deller T, Staufenbiel M, Mathews PM, Jucker M. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science. 2002 Nov 15;298(5597):1379. PubMed.
- Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 2003 Apr;9(4):448-52. PubMed.
- Ferrer I, Boada Rovira M, Sánchez Guerra ML, Rey MJ, Costa-Jussá F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol. 2004 Jan;14(1):11-20. PubMed.
- Masliah E, Hansen L, Adame A, Crews L, Bard F, Lee C, Seubert P, Games D, Kirby L, Schenk D. Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease. Neurology. 2005 Jan 11;64(1):129-31. PubMed.
- Brendza RP, Bacskai BJ, Cirrito JR, Simmons KA, Skoch JM, Klunk WE, Mathis CA, Bales KR, Paul SM, Hyman BT, Holtzman DM. Anti-Abeta antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice. J Clin Invest. 2005 Feb;115(2):428-33. PubMed.
- Fryer JD, Taylor JW, Demattos RB, Bales KR, Paul SM, Parsadanian M, Holtzman DM. Apolipoprotein E markedly facilitates age-dependent cerebral amyloid angiopathy and spontaneous hemorrhage in amyloid precursor protein transgenic mice. J Neurosci. 2003 Aug 27;23(21):7889-96. PubMed.
- Agadjanyan MG, Ghochikyan A, Petrushina I, Vasilevko V, Movsesyan N, Mkrtichyan M, Saing T, Cribbs DH. Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J Immunol. 2005 Feb 1;174(3):1580-6. PubMed.
- Lemere CA, Beierschmitt A, Iglesias M, Spooner ET, Bloom JK, Leverone JF, Zheng JB, Seabrook TJ, Louard D, Li D, Selkoe DJ, Palmour RM, Ervin FR. Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet. Am J Pathol. 2004 Jul;165(1):283-97. PubMed.
- Solomon B. Generation of anti-beta-amyloid antibodies via phage display technology towards Alzheimer's disease vaccination. Vaccine. 2005 Mar 18;23(17-18):2327-30. PubMed.
- Herzig MC, Winkler DT, Burgermeister P, Pfeifer M, Kohler E, Schmidt SD, Danner S, Abramowski D, Stürchler-Pierrat C, Bürki K, van Duinen SG, Maat-Schieman ML, Staufenbiel M, Mathews PM, Jucker M. Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis. Nat Neurosci. 2004 Sep;7(9):954-60. PubMed.
Sorrento: Scientists Stop Clubbing, Start Tweaking, γ-Secretase
One of the surprises at the AD/PD 2005 conference held last week in Sorrento, Italy, lay in just how many groups presented new experimental therapy approaches in various stages of preclinical development. This welcome news accompanies the arrival of a constant stream of new mouse strains that model aspects of Alzheimer disease. Even with their limitations, these models will enable investigators to test their favorite fledgling therapy in a battery of systems that together will be more physiologically meaningful than the cell-free or cell-based assay that generated the original idea or compound. Many of those treatment approaches have in common that they try to reduce production of the Aβ peptide, considered a culprit in AD pathogenesis, by choking the γ-secretase complex, and some news on that effort is summarized here. For presentations focused on β- and α-secretase, see companion Sorrento story.
γ-Secretase Modulators: The New Wave?
Steve Wagner at the Californian biotechnology company TorreyPines Therapeutics introduced a series of new compounds. In the past few years, it has become clear that certain non-steroidal anti-inflammatory drugs (NSAIDs) lower levels of Aβ40/42 in a way that has little do to with their ostensible targets, COX1/2 (see ARF Live Discussion). These NSAIDs subtly alter the γ-secretase complex that produces Aβ in such a way that the enzyme spits out less of the pathogenic peptide while continuing apace with its necessary job of cutting a growing list of other, physiological substrate proteins (see ARF related news story). Once this idea of so-called allosteric modulation of the γ-secretase enzyme complex was in the air, the hunt was on for compounds that pull off this trick more potently than do NSAIDs. That is because most NSAIDs are quite toxic at the doses required for meaningful Aβ42 lowering. (In recent months, troubles about cardiovascular side effects have led to market withdrawal, more restrictive labeling, and lower dosing recommendations for some NSAIDS for arthritis treatment. This raises doubts about whether they'll ever be approved for AD prevention, as prevention requires a greater safety margin than does the treatment of chronic pain, see ARF related news story, see ARF related news story).
Wagner's team appears to have made a catch with a series of small molecules called NGX 83232 and another compound called NGX96256. In Sorrento, Wagner explained his team's efforts to screen for chemicals that lower Aβ42 production while weeding out those that also lower production of NICD. This is the intracellular tail of the γ-secretase's best studied other substrate, Notch, and it is also the protein that caused the downfall of first-generation γ-secretase inhibitors. These mechanism-based inhibitors went into clinical trials, including a phase 2 trial by Bristol Myers Squibb, where they proved too toxic (see more in May section below.) From Wagner's screen, however, one compound emerged that looked promising, and medicinal chemists fashioned 650 derivatives of it until they found some that did the job in the single-digit or sub-nanomolar concentration ranges drug developers like to use.
In addition to sparing Notch, the NGX 83232 compounds also leave unchanged levels of AICD, the cytoplasmic tail of APP. This peptide is widely thought to enter the nucleus for signaling, and recently turned out to regulate the production of none other than its parent APP in a positive, auto-regulatory feedback loop (von Rotz et al., 2004, and confirmed independently in Sorrento with a poster presentation by Sébastien Hébert in Bart de Strooper's laboratory.)
The NGX compounds did not change the total amount of Aβ produced, but they did shift the relative amounts of its various forms. There was less of the pathogenic Aβ 42 and 40 and more of its shorter forms having 37 and 38 amino acids. Little is known about these smaller, less fibrillogenic versions of Aβ, but scientists presume them to be relatively harmless.
Wagner reported that the compounds showed this effect in human neuroblastoma cells overexpressing APP and in mixed brain cultured from Tg2576 mice. Largely the same result appeared in plasma and brain extracts from Tg257 mice fed the compounds for three days. Wagner noted that the compounds did not affect the normal cleavage of ε-cadherin and ErbB4, which are two of the other physiological substrates of γ-secretase. They also did not disturb ε cleavage of APP, which is thought to precede the γ-cleavage that then releases Aβ. In response to a question, Wagner noted that his group tried NSAIDs but was unable to find a clear effect without toxicity.
(Editor's note added on March 21, 2005: This week's biotech newsletter BioCentury reported that the German/British biotechnology company Cellzome has signed an agreement with the pharmaceutical company Johnson and Johnson to develop its own small-molecule γ-secretase modulator.)
γ-Secretase Inhibitors: Down But Not Out
γ-secretase has been a favorite drug target throughout the 1990s, even before researchers identified presenilin as its catalytic component and later fingered three other members of the complex. Yet when the list of the enzyme's other substrates grew and notch-related side effects appeared, its luster dimmed and it became de rigeur among academic scientists to pronounce it dead as a drug target. In Sorrento, Patrick May of Eli Lilly and Company in Indianapolis, a veteran in the search of γ-secretase inhibitors, contended otherwise, saying newer compounds are still worth pursuing.
May recounted the history of the long and arduous search for such inhibitors, saying the work started with what by today's standards are unsophisticated tools and little supporting knowledge about just how complex this enzyme and the flux of Aα through the body is. "We were working in the dark in those days," he told the audience. Early successes with an inhibitor called DAPT made the goal look attainable, especially since the compounds appeared to have delayed pharmacodynamic effects on Aβ levels and amyloid deposition after the drug had left the mice's body (Dovey et al., 2001).
Next, Lilly scientists made the LY series of inhibitors, which are largely unpublished except for LY411575. Merck and Pfizer have studied such compounds, as well (see Best et al., 2005, Lanz, 2004), and some now act at picomolar doses without apparent cytotoxicity, May said. Yet it is also known that at least LY411575 at higher doses ravages the immune system and intestine of mice (Wong et al, 2004) The side effects have raised the question of whether one can achieve a clinically significant reduction in Aβ while otherwise preserving essential function of regulated intramembrane cleavage. This issue of the therapeutic window can only be answered in the clinic, May said.
Toward that end, May talked about the compound LY 450139, which inhibits γ-secretase less potently but also has a milder effect on Notch cleavage. "We want to gingerly go into the clinic with this second generation," May said. Long-term administration to beagle dogs went well, and the compound reduced CSF Aβ levels. LY450139 is now in preparation for use in clinical biomarker studies, May said. A randomized phase 1 trial of 70 AD patients taking the drug for six weeks has helped the company approximate an apparently safe dose, but otherwise was inconclusive. That is largely because lumbar CSF levels of Aβ did not drop as markedly as was hoped, and an observed Aβ drop in plasma levels is difficult to interpret at this point. A large efficacy trial, therefore, is not imminent, May concluded, as the company may have to try out a slightly higher dose first. Other companies developing g-secretase inhibitors include Bristol-Myers Squibb, Novartis, Amgen, Sanofi-Aventis, but they have not made data public.—Gabrielle Strobel.
- Sorrento Secretase News: Baiting β, Awakening α
- FRETting Pays off—NSAIDs Target Presenilins, Reduce Aβ42
- NSAIDs and AD—Prevention Better Than Cure?
- Safety Concerns over Galantamine; Fallout from Rofecoxib Debacle Grows
- von Rotz RC, Kohli BM, Bosset J, Meier M, Suzuki T, Nitsch RM, Konietzko U. The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor. J Cell Sci. 2004 Sep 1;117(Pt 19):4435-48. PubMed.
- Dovey HF, John V, Anderson JP, Chen LZ, de Saint Andrieu P, Fang LY, Freedman SB, Folmer B, Goldbach E, Holsztynska EJ, Hu KL, Johnson-Wood KL, Kennedy SL, Kholodenko D, Knops JE, Latimer LH, Lee M, Liao Z, Lieberburg IM, Motter RN, Mutter LC, Nietz J, Quinn KP, Sacchi KL, Seubert PA, Shopp GM, Thorsett ED, Tung JS, Wu J, Yang S, Yin CT, Schenk DB, May PC, Altstiel LD, Bender MH, Boggs LN, Britton TC, Clemens JC, Czilli DL, Dieckman-McGinty DK, Droste JJ, Fuson KS, Gitter BD, Hyslop PA, Johnstone EM, Li WY, Little SP, Mabry TE, Miller FD, Audia JE. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem. 2001 Jan;76(1):173-81. PubMed.
- Best JD, Jay MT, Otu F, Ma J, Nadin A, Ellis S, Lewis HD, Pattison C, Reilly M, Harrison T, Shearman MS, Williamson TL, Atack JR. Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-di. J Pharmacol Exp Ther. 2005 May;313(2):902-8. PubMed.
- Lanz TA, Hosley JD, Adams WJ, Merchant KM. Studies of Abeta pharmacodynamics in the brain, cerebrospinal fluid, and plasma in young (plaque-free) Tg2576 mice using the gamma-secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]az. J Pharmacol Exp Ther. 2004 Apr;309(1):49-55. PubMed.
- Wong GT, Manfra D, Poulet FM, Zhang Q, Josien H, Bara T, Engstrom L, Pinzon-Ortiz M, Fine JS, Lee HJ, Zhang L, Higgins GA, Parker EM. Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem. 2004 Mar 26;279(13):12876-82. PubMed.
Sorrento Secretase News: Baiting β, Awakening α
Spooked by the pitfalls of γ-secretase drug development (see companion Sorrento story), BACE fans like to point out that their aspartyl protease may be a safer target. They take comfort in pointing out that compared to γ-secretase knockout mice, BACE knockout mice are doing just fine, thank you. (BACE cleaves APP before γ-secretase, and is therefore the first target in the pathogenic pathway of APP cleavage.) What's more, last week at the AD/PD 2005 conference in Sorrento, Sukanto Sinha of Elan suggested that if a safe BACE inhibitor could be found, restraining this protease slightly might be all that's needed for an AD treatment. The Elan team crossed PDAPP (i.e. APP-overexpressing) mice to heterozygous BACE knockout mice in order to reduce their BACE levels by half. These crosses have a mere 10 percent dent in their brain Aβ levels compared to PDAPP mice, yet show marked reductions in their soluble Aβ, amyloid deposition, and dystrophic neurites, Sinha reported.
What of it, then? Where are the inhibitors? Years of drug discovery effort have shown how difficult it is to try to fit small-molecule inhibitors into the inhospitable plane that is this enzyme's active site. Peptide inhibitors, such as those described in Sorrento by Jordan Tang, of the Oklahoma Medical Research Foundation in Oklahoma city, rarely become drugs, though they have been useful research tools in guiding structural studies and inhibitor design. Finally, small-molecule candidates are coming out and two are rumored to have entered phase 1 studies. Other, purely experimental, compounds have been reported in the literature (see e.g. Lefranc-Jullien et al., 2005, Hu et al. 2004, Hom et al., 2004.)
In Sorrento, Adam Simon of Merck Research Laboratories in West Point, Pennsylvania, reported on one promising molecule, dubbed Merck3. Simon noted that the present compound is selective for BACE1, inhibiting neither BACE 2 nor renin, which had been a problem with prior compounds (see also Stachel et al., 2004). The scientists tested this molecule in a mouse strain made by Bruce Lamb to express full-length human wild-type APP from a yeast artificial chromosome. They pumped the compound, which enters cells but appears not to cross the BBB effectively, into a brain ventricle and then measured Aβ40 and 42 levels. One hopes that Merck is developing more bioavailable compounds internally.
The Merck scientists also measured the APP fragment sAPPalpha, the product of the α-secretase, to test the underlying assumption that inhibiting BACE would shift APP processing toward the non-amyloidogenic α-secretase cleavage. At one, seven, and 14 days, the researchers saw a drop in Aβ42 and a concomitant increase in sAPPalpha in brain homogenates. This result suggests that α-secretase and BACE compete for APP in vivo, and it dovetails with a recent demonstration that turning up alpha-secretase even by a notch can reduce amyloid deposition and improve learning in APP-transgenic mice (Postina et al., 2004), Simon added.
Less Aα, More sAPPalpha: Buy One, Get One Free?
The goal of shifting APP cleavage away from β- and γ-secretase and toward α-secretase emerged as the field's hot new trail at the Sorrento meeting. Such a shift might yield the added bonus that the sAPPalpha fragment not only precludes the making of Aβ, but also does good in its own right. Various studies over the years have sung its praises for being neurotrophic, as well as for protecting against Aβ-induced LTP deficiency and against learning deficits induced by anti-muscarinic agents. In Sorrento, many informal conversations as well as formal presentations focused on it, most prominently Abraham Fisher's lecture reviewing the AF267B compound (previously covered in Alzforum story). Below is a summary of one other such approach, by Falk Fahrenholz of University of Mainz, Germany.
Fahrenholz believes ADAM-10 is one of several endogenous α-secretases, ADAM-17 being another. His group's paper last year (Postina et al., 2004) had raised the prospect of upregulating ADAM-10, and in Sorrento, he presented initial data on how one could go about it. Already, some γ-protein coupled receptors and their subsequent signaling pathways are known to control α-secretase expression. Certain non-tumorigenic protein kinase C activators, such as bryostatin, are reported to boost sAPPα levels (Etcheberrigaray et al., 2004). Fisher's M1-selective agonists do so, as well, by activating PKC molecules that are anchored to M1 acetylcholine receptors, Fisher said.
Another means of activating α-secretase could be found in endogenous hormones that modulate signal transduction pathways upstream of α secretase, Fahrenholz said. He is investigating the pituitary adenylate cyclase-activating polypeptide (PACAP) and its γ-protein coupled receptor PAC1. PACAP and its receptors occur in the human cerebral cortex and hippocampus and are implicated in neuroprotection, Fahrenholz noted. His most recent data show that in neuroblastoma cells expressing PACAP receptors, the hormone increases sAPPα levels at nanomolar doses, whereas an ADAM-10 inhibitor prevented the PACAP-induced α-secretase activity. Further dissection of the pathway confirmed the role of MAP kinase, protein kinase C, PiP3 and other players, Fahrenholz said, adding that he has begun testing PACAP in a mouse model.
Fahrenholz is hopeful this hormone could become a drug because it is known to reach the brain (for a review of PACAP transport, see Dogrukol-Ak et al. 2004.) Incidentally, PACAP is also thought to stimulate proliferation of neural stem cells in adult mouse brain (Mercer et al.,). Alternatively, other known PAC receptor agonists might work, or else, combing the ADAM-10 promoter for binding sites of other transcription factors that can be regulated by outside signals might prove fruitful. Finally, low cellular cholesterol appears to encourage α cleavage of APP, suggesting one mechanism by which statin treatment may help stave off AD, Fahrenholz concluded.—Gabrielle Strobel.
- Sorrento: Scientists Stop Clubbing, Start Tweaking, γ-Secretase
- San Diego: Treating Forgetfulness—Triple Transgenics Provoke
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- Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M, Flamez P, Dequenne A, Godaux E, Van Leuven F, Fahrenholz F. A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest. 2004 May;113(10):1456-64. PubMed.
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- Mercer A, Rönnholm H, Holmberg J, Lundh H, Heidrich J, Zachrisson O, Ossoinak A, Frisén J, Patrone C. PACAP promotes neural stem cell proliferation in adult mouse brain. J Neurosci Res. 2004 Apr 15;76(2):205-15. PubMed.
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Sorrento: More Fun, Less Amyloid for Transgenic Mice
If you need a reason to get off the bench and work up a sweat, consider this: Increasing physical and mental activity dramatically lowers amyloid plaque deposition in a mouse model of Alzheimer's disease, according to a study presented last week by Sangram Sisodia from the University of Chicago at the AD/PD 2005 conference in Sorrento, Italy. Young mice given access to exercise wheels and toys showed changes in the expression of genes implicated in learning and memory, blood vessel growth, neurogenesis, and cell survival pathways. Moreover, Aβ-degrading enzymes appeared to be more active in the exercising mice. The results, which were published in the March 11 issue of Cell, provide a biochemical link between environment and Alzheimer's progression by showing that a modest change in the experience of young animals can decrease amyloid levels.
Exercise, whether mental or physical, seems to fend off Alzheimer's disease in humans (Teri et al., 2003). Likewise, other studies with adult AD mice and normal aging beagles have shown that switching the animals from standard-issue laboratory cages to surroundings where they can exercise and play improves cognitive function (Milgram et al., 2005; Arendash et al., 2004.). Also in Sorrento, researchers in Carl Cotman's group at the University of California, Irvine, reported that five months of voluntary exercise stimulated both neurogenesis and learning in the water maze of CRND8 APP-transgenic mice. These mice, too, had fewer amyloid deposits in their cortex and hippocampus, as well as lower brain Aβ levels.
To look for possible effects on amyloid metabolism early in life, first author Orly Lazarov and colleagues started with weanling AD mice, treating the animals to daily sessions in a large cage with running wheels, brightly colored tunnels, and toys. They collaborated with researchers from Stanford University, the University of Kentucky, and the University of Pittsburgh, to measure Aβ levels, plaque deposition, and gene expression profiles between the enriched mice and those that were left in standard laboratory cages.
Their results showed that young mice that were allowed to frolic had decreased levels of Aβ and fewer plaques. The stimulated mice displayed a two-fold higher activity of the Aβ-degrading enzyme neprilysin, suggesting they were better able to clear Aβ than the normally housed mice. In the group of six enriched mice, the three who spent the most time on the running wheel had the lowest levels of Aβ. It remains to be determined whether clearer brains are the cause or the result of the mice's propensity to exercise. At this point, the Cotman group ascribes the anti-amyloid effect they have observed more to reductions in APP processing than to increases in neprilysin and IDE. Follow-up studies will sort out this question.
In Sisodia's study, microarray analysis of hippocampal gene expression identified enrichment-specific changes in transcript level for genes involved in a variety of pathways including Aβ sequestration and cell survival. Some of the enriched animals showed upregulation of genes thought to protect against neurodegeneration, such as BDNF, and other transcripts known to be increased by exercise.
This study involved a small number of mice and no physiological or behavioral tests were performed, so it remains to be shown how the changes in plaque burden and gene expression relate to cognitive status. Also, it is not clear what aspects of a complex natural environment are replicated by running wheels and colored toys for caged mice. "The demonstration that environmental factors can alter Aβ deposition raises more questions than it answers," write Stanislav Karsten and Daniel Geschwind of UCLA in an accompanying preview. But, they say, the study opens a door to understanding the interplay of genetics and the environment in Alzheimer's disease.—Pat McCaffrey, Gabrielle Strobel
Pat McCaffrey is a science writer in Newton, Massachusetts
- Teri L, Gibbons LE, McCurry SM, Logsdon RG, Buchner DM, Barlow WE, Kukull WA, LaCroix AZ, McCormick W, Larson EB. Exercise plus behavioral management in patients with Alzheimer disease: a randomized controlled trial. JAMA. 2003 Oct 15;290(15):2015-22. PubMed.
- Milgram NW, Head E, Zicker SC, Ikeda-Douglas CJ, Murphey H, Muggenburg B, Siwak C, Tapp D, Cotman CW. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study. Neurobiol Aging. 2005 Jan;26(1):77-90. PubMed.
- Arendash GW, Garcia MF, Costa DA, Cracchiolo JR, Wefes IM, Potter H. Environmental enrichment improves cognition in aged Alzheimer's transgenic mice despite stable beta-amyloid deposition. Neuroreport. 2004 Aug 6;15(11):1751-4. PubMed.
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Sorrento: They’ve Just Got to Have It—Where’s That Biomarker? Part 1
With heart disease, high cholesterol levels prompt the doctor to prescribe statins and urge other preventive measures. A suspicious PSA screens warns the doctor that an aging prostate is misbehaving. Why is such a laboratory test not available for AD?
It’s not for lack of trying, say scientists. The search for biomarkers has been difficult. Early candidates have fallen by the wayside for lack of robustness, sensitivity, or specificity, even as the advent of new experimental therapies has made the need for a reliable test more pressing. Researchers believe that new therapies will show the most promise for the earliest stages of AD, which may precede overt symptoms by years. For this reason, that elusive, telltale giveaway that the underlying disease process is gnawing away at a person’s brain has become a coveted prize for both practicing clinicians and clinical trial researchers. The 7th AD/PD 2005 conference held March 9-13 in Sorrento offered the latest news on some of the ongoing efforts to finger such a marker for AD, and budding efforts in Parkinson disease (PD). Here are selected highlights.
John Growdon of Massachusetts General Hospital in Boston said that all too often, molecules that come recommended by cellular or epidemiological science sadly disappoint when tested for their potential as predictive biomarkers. For example, ApoE is a genetic risk factor that makes for a poor biomarker. The latest case in point is homocysteine, which boasts a host of data implicating it in age-related neurodegeneration in general, and in Parkinson disease in particular (see Alzforum live discussion). However, a human study at MGH showed that measuring its levels does not enable a clinician to distinguish AD from Mild Cognitive Impairment (MCI), PD, or normal controls. Levels did correlate with age, levodopa use, and cognitive impairment in PD, and they were low in people who took multivitamins. This further strengthens a growing literature suggesting that dietary folate supplements may be a helpful component of PD treatment and prevention, but it also means that homocysteine will not become a biomarker, Growdon said.
In Alzheimer's, the seemingly obvious approach of turning Aβ levels into a biomarker has been anything but straightforward. Doctors would most like to have a blood test, as patients more readily roll up a sleeve than curl up sideways for a spinal tap. But no one to date has found Aβ differences in serum that robustly predict the patient’s condition. Growdon added another downer to this hope by reporting that in a plasma Aβ40/42 study conducted at MGH, mean Aβ40/42 levels did not differ among MCI, AD, or control patients. Plasma Aβ levels did rise with age, but were not related to AD history, severity, or acetylcholinesterase medication. “For sporadic AD, plasma Aβ itself is not likely to be a useful biomarker,” Growdon said.
Even so, blood-based detection of amyloid-related markers is far from dead, at least when approached from a different angle. For example, researchers led by Andre Delacourte at INSERM in Lille, France, are trying to link to AD changes in certain C-terminal fragments of APP, as well as what they believe are pathological fragments of Aβ found in lymphocytes and platelets.
It’s All in the Spine
How about cerebrospinal fluid, then? Generally speaking, the invasive nature of a spinal tap has made American clinicians more reluctant to perform it than is the case in Europe, where CSF biomarker research has long been actively pursued. Kaj Blennow presented a summary of such work at the University of Goteborg in Sweden. Blennow noted that more than 45 biomarker studies have documented a large increase of CSF total tau (T-tau) in AD. However, the sensitivity (how many cases does it pick out?) and especially the specificity (how well does it distinguish between similar conditions?) of this measure limits its use as a diagnostic marker. CSF Aβ42 levels decrease with AD, but its specificity, too, is insufficient.
By contrast, phosphorylated tau (P-tau) discriminates better between related disorders and will supersede total tau as a marker, Blennow said. Helping put to rest a debate about which of tau’s phosphorylation epitopes are best, Blennow reported that in a recent study, three different tau methods yielded the same results when compared in material from the same patients. The ability of the combination of T-tau, P-tau, and Aβ42 to predict who among a group of 52 MCI cases would progress to full-blown AD in two years reached a respectable 94 percent, Blennow reported. Furthermore, a study asking whether known CSF markers can predict who among a group of 54 healthy population-based people would develop AD found that both T-tau and Aβ42 were significantly changed as early as eight years prior to diagnosis; a community-based study yielded the same result. In a separate presentation, Mony de Leon, from New York University, reported similar results from his MCI study, concluding that the increase in CSF P-tau and decrease in Aβ42 can sharpen the diagnosis at early stages. The main limitation of these markers is that they differentiate AD poorly from other forms of dementia, Blennow said.
Another important part of putting a putative biomarker through its paces involves testing whether it can identify the biochemical effect of a drug. Blennow reported that in AD patients who took the known acetylcholinesterase inhibitors donepezil or galantamine for 6 to 12 months, the researchers detected a marked increase in CSF acetylcholinesterase activity with donepezil, and a smaller increase with galantamine. Neither tau nor Aβ levels changed during this drug treatment, confirming again that these drugs improve symptoms but do not change the disease process.
What about experimental treatments? CSF tau and Aβ levels did not budge, either, after treatment with clioquinol, a drug that disrupts metal-Aβ complexes (see Alzforum live discussion ). Nor did they change after up to 12 months of treatment with simvastatin or atorvastatin, Blennow said. This latter finding was a bit disheartening, because a small previous study had been able to detect a dent in CSF levels of Aβ40 in people with mild AD. Yet even when Blennow’s group divided the subjects in his small study into mild and moderate cases, CSF Aβ and tau stayed flat. “We could not reproduce that earlier trend,” Blennow said (see Simons et al., 2002, Sjogren et al., 2003).
Faced with these stubbornly flat levels of CSF Aβ and tau after all these different treatments, Blennow asked if anything could nudge them up or down. Was the problem in the method? Apparently not, because in 15 people who sustained a head trauma, Aβ rose dramatically after four days and fell down again after 10 days. “So we can detect these changes but have not yet found a treatment that affects Aβ CSF levels,” Blennow concluded.
Diagnosis by Committee: Biomarker Panels
Surely, there must be other biomarkers that perhaps would nail a diagnosis when combined with P-tau or Aβ in a larger panel? One such candidate might be found around oxidative enzyme reactions in the CSF, reported Domenico Pratico of the University of Pennsylvania in Philadelphia. Practico introduced 12/15 lipoxygenase as a member of an enzyme family that inserts molecular oxygen into polyunsaturated fatty acids and therefore represents an enzymatic source of oxidative products, rather than the free radicals commonly blamed for oxidative stress. After showing that relevant, amyloid-bearing brain regions of postmortem AD tissue had an increase in protein level and activity of this enzyme (Pratico et al., 2004), the researchers began exploring its potential as a future biomarker in living people with AD. In a study of 15 people with AD, 10 with MCI, and 20 controls, CSF levels of a biochemical 12/15 LOX readout were lowest in controls, higher in MCI cases, and yet higher in AD. This new data correlated with more established measures of oxidation, such as isoprostanes, and also with the expected Aβ42 decrease and tau increase in CSF, Pratico said (for review, see Pratico, 2005).
Blennow described his group’s attempt at finding new members for a panel of biomarkers as focusing, in part, on a multiplex immunoassay called Luminex xMAP. Researchers coat microspheres with antibodies to several different putative biomarkers, and then use laser excitation and fluorescence to quantify the signal. Testing the method, Blennow’s group covered microspheres with antibodies to tau, Aβ42, and P-tau, and found that results in a first human study resembled ELISA in sensitivity and specificity. His group is now using the technique to explore marker cocktails including N-terminal Aβ fragments, Aβ oligomers, synaptic proteins, cytokines, growth factors, and other proteins.
Several groups are performing CSF proteomics using surface-enhanced laser desorption ionization time-of-flight (Seldi-Tof) mass spectrometry to look for marker patterns. Blennow noted that a collaboration of Swedish, Danish, and Finnish groups has identified some 30 components in peaks of increased and decreased proteins among 98 early AD patients, 31 FTD patients, and 49 controls. The component proteins include transthyretin, cystatin C (which has been implicated in AD since the 1980s, most recently by Cathcart et al., 2005), secretogranin, chromogranin, neuroendocrine protein, β 2 microglobulin, various oxidative stress proteins, and others. Individually, none of these predicts AD better than Aβ or P-tau, but in the right combination they may well, Blennow said.
Gene expression microarrays and proteomics also serve to search for a genetic fingerprint of PD. Growdon and Peter Riederer at the University of Wuerzburg, Germany, both summarized such studies. Growdon mentioned initial data of an ongoing blood-based study of 100 idiopathic PD patients, which has to date identified genes of cellular quality control pathways, mitochondrial genes, and others. Likewise, Riederer’s group has begun a proteomics study with PD patients who donate a blood sample every six months for three years.
In addition, Riederer summarized a microarray study performed with Moussa Youdim at Technion in Haifa, Israel, in which the scientists used substantia nigra tissue samples from sporadic PD patients to establish two groups of about 70 genes whose expression went either up or down in that brain region. Standing out from among the genes whose expression dropped was SKP1A, which is part of an E3 ubiquitin ligase complex in substantia nigra and could help explain proteasome dysfunction in PD. Another gene whose expression went down (and which also comes with a biological rationale), is aldehyde dehydrogenase. It functions in dopamine metabolism. These and other genes are being investigated in blood samples of PD patients and healthy people to see whether any of them could pass muster as future biomarkers, said Riederer (see also Grunblatt et al., 2004).—Gabrielle Strobel.
Q&A with Kaj Blennow
Q: I would expect CSF AChE to decrease after treatment with AChE inhibitors?
A: This increase is a mystery. When we performed our study, our hypothesis was to find a decrease, but we found the reverse. We do not have a good explanation, but this finding has been replicated by other groups (Parnetti et al., 2002). The best guess today is that it is a compensatory upregulation.
Q: With such unexpected results, what ‘s the upshot of your CSF drug studies?
A: The main conclusion from our study is that using CSF,
1) it is possible to identify and monitor a biochemical effect of a drug;
2) it is possible to identify differences between drugs with different modes of action (donepezil vs. galantamine);
3) the changes in CSF are dose-dependent;
4) the changes correlate with the clinical effect of the drug.
Q: What’s the future of CSF analysis?
A: We hope that CSF analyses can be of value to identify and monitor the biochemical effect of new types of treatment, such as BACE1 inhibitors or Aβ vaccination regimes. We also think that there may be two types of CSF biomarkers for BACE1 inhibitors and Aβ vaccination:
1) "Primary" or "Specific" biomarkers based on the mode of action, i.e., Aβ42, Aβ40, α-sAPP and β-sAPP to directly monitor Aβ metabolism, but also
2) "Secondary" or "downstream" CSF biomarkers, e.g., CSF T-tau (or other markers for neuronal degeneration such as NSE), to monitor if these drugs also will slow down the degenerative process.
- Simons M, Schwärzler F, Lütjohann D, von Bergmann K, Beyreuther K, Dichgans J, Wormstall H, Hartmann T, Schulz JB. Treatment with simvastatin in normocholesterolemic patients with Alzheimer's disease: A 26-week randomized, placebo-controlled, double-blind trial. Ann Neurol. 2002 Sep;52(3):346-50. PubMed.
- Sjögren M, Gustafsson K, Syversen S, Olsson A, Edman A, Davidsson P, Wallin A, Blennow K. Treatment with simvastatin in patients with Alzheimer's disease lowers both alpha- and beta-cleaved amyloid precursor protein. Dement Geriatr Cogn Disord. 2003;16(1):25-30. PubMed.
- Praticò D, Zhukareva V, Yao Y, Uryu K, Funk CD, Lawson JA, Trojanowski JQ, Lee VM. 12/15-lipoxygenase is increased in Alzheimer's disease: possible involvement in brain oxidative stress. Am J Pathol. 2004 May;164(5):1655-62. PubMed.
- Praticò D. Peripheral biomarkers of oxidative damage in Alzheimer's disease: the road ahead. Neurobiol Aging. 2005 May;26(5):581-3. PubMed.
- Cathcart HM, Huang R, Lanham IS, Corder EH, Poduslo SE. Cystatin C as a risk factor for Alzheimer disease. Neurology. 2005 Feb 22;64(4):755-7. PubMed.
- Grünblatt E, Mandel S, Jacob-Hirsch J, Zeligson S, Amariglo N, Rechavi G, Li J, Ravid R, Roggendorf W, Riederer P, Youdim MB. Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes. J Neural Transm. 2004 Dec;111(12):1543-73. PubMed.
- Amici S, Lanari A, Romani R, Antognelli C, Gallai V, Parnetti L. Cerebrospinal fluid acetylcholinesterase activity after long-term treatment with donepezil and rivastigmina. Mech Ageing Dev. 2001 Nov;122(16):2057-62. PubMed.
Sorrento: They’ve Just Got to Have It—Where’s That Biomarker? Part 2
This is part 2 of the biomarker update from Sorrento.
Besides biochemistry and genetics, the search for early markers has focused heavily on brain imaging. Pittsburgh Compound-B (PIB) has broken new ground by lightening up amyloid deposits in the brain of AD patients. It is also said to distinguish better between AD and related dementias than does 18F-FDG PET, another imaging method that has shown promise and can inform the diagnosis at specialized medical centers. To become widely available, safer, and cost-effective, PIB needs to be coupled to a ligand other than 11C; alternatively, MRI-based tracers may be developed (see ARF related news story). For now, however, PIB continues to lead a growing pack of experimental amyloid dyes into uncharted human territory. The Alzheimer Research Forum has covered the testing of PIB extensively (see ARF recent conference update). Below is additional news from Sorrento.
Agneta Nordberg of the Karolinska Institute in Stockholm, Sweden, heads the group that began the first prospective human study of PIB at the PET Center in Uppsala. In Sorrento, she presented data of a follow-up measurement of a group of 16 AD patients 1.5 to 2.5 years after their first PIB imaging session. Patients whose cognitive scores declined during this time showed an increase in PIB retention at the second measurement, while those whose cognition stayed stable also had largely stable PIB retention. People whose cognition worsened over the course of this time started the study with higher PIB binding values than did people who remained stable. Together, this suggests that PIB retention and, by extension, amyloid load is correlated with performance and that amyloid deposition precedes the clinical progression of AD, said Nordberg, but she added that her speculative interpretation needs more data and statistics to back it up. Nordberg said she would reveal new data on the small prospective MCI cohort in the study when that group contained at least 20 people. The burning question there is whether PIB can predict who among those with a diagnosis of MCI will develop AD, and when. “We are still at the beginning of this research. New ligands will come up and new methods to evaluate them,” concluded Nordberg.
Chris Rowe preceded his presentation of initial data from his group’s PIB studies by deploring that the commercial licensing agreement for PIB has made it more difficult for academic scientists to test PIB more broadly. To date, only five out of many more qualified academic PET centers have joined the Swedish teams and the original Pittsburg team in testing PIB, said Rowe. They are in Toronto, London, Turku/Finland, Washington University in St. Louis, Missouri, and Austin Hospital in Heidelberg, Australia.
Working at this latter center on the outskirts of Melbourne, Rowe collaborated with others at Monash University and the University of Melbourne to start a prospective study comparing MRI, FDG-PET and PIB-PET in people with AD, DLB, and controls. A neuropsychiatric test battery, as well as plasma Aβ and ApoE genotyping, are part of the study. Five patients with mild AD who have been imaged to date all have abnormal PIB scans with high PIB retention in the cortex, said Rowe. Of the five control subjects, four show low PIB binding. A 78-year-old woman, who complained about being “less sharp” than before but has an abnormally low score only in one verbal fluency test, retained moderate amounts of PIB in her cortex, said Rowe.
Four patients enrolled so far into the LBD group all have advanced dementia and show PIB retention in a pattern that appears distinct from that of AD, said Rowe. LBD patients have lower overall uptake levels than do AD patients at the same stage of disease. Moreover, they show more diffuse retention, with a significant PIB signal in areas that are relatively spared in AD, such as the occipital lobe and primary sensory and motor cortex. By testing PIB and FDG-PET side-by-side, this study, as well as Nordberg’s, will try to find out whether amyloid PET or PET based on glucose metabolism is more sensitive at picking up early stages of AD.
No matter who wins this particular contest, both methods require that the patients agree to flood their veins with radioactive tracers for several hours. Researchers are trying to find other methods that dispense with this infusion. In Sorrento, Norbert Schuff of the University of California, San Francisco, introduced early data on arterial spin-labeling perfusion MRI, which lights up under-perfused brain areas without an injection. Schuff suggested that once the signal-noise ratio of this method has improved, it, too, could help the clinician perform a differential diagnosis, because it generated different anatomical patterns of insufficient perfusion between related neurodegenerative conditions (see also Johnson et al., 2005). A broad-based multinational initiative has begun to validate the best among the multitude of promising approaches that neuroimaging has produced in recent years. Check back tomorrow for a news story on this big-science slice of the broader biomarker story.—Gabrielle Strobel.
- Visualizing Success with MRI of Amyloid Plaques in Live Mice
- Philadelphia: All Eyes on PIB Imaging—Is It Coming Along?
- Johnson NA, Jahng GH, Weiner MW, Miller BL, Chui HC, Jagust WJ, Gorno-Tempini ML, Schuff N. Pattern of cerebral hypoperfusion in Alzheimer disease and mild cognitive impairment measured with arterial spin-labeling MR imaging: initial experience. Radiology. 2005 Mar;234(3):851-9. PubMed.
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Sorrento: Sorting Out Shedding of Ectodomains
Not only the sweet scent from the ubiquitous citrus trees aroused the senses at the 7th international AD/PD meeting in Sorrento, but the unlikely theme of ectodomains managed to do so, too. And while gravity’s role in shedding ripe fruit may be obvious, we now know that sorting proteins have their own particular gravitas when it comes to ectodomain shedding, particularly those of the amyloid-β precursor protein (AβPP). What are sorting proteins anyway, you may well ask? Read on…
Olav Andersen from Thomas Willnow’s lab at the Max Delbrueck Center for Molecular Medicine, Berlin, revealed that SorLA, a member of the LDL receptor family, may play a major role in regulating the processing of AβPP.
Andersen proposed that SorLA may be more of a sorting receptor than an apolipoprotein receptor. First, the N-terminal of SorLA contains a motif that shares homology to the well-characterized yeast VPS10 intracellular sorting receptor. Second, only 10 percent of SorLA is found on the cell surface, which would seem to preclude a major lipoprotein receptor role; the majority of the protein resides in the Golgi apparatus. And third, the C-terminal of the protein contains a motif that facilitates binding to the TGA family of proteins, which facilitates protein sorting. But it might have been a report last year showing loss of SorLA, also called LR11, in Alzheimer disease (AD) neurons (see Scherzer et al., 2004) that piqued Andersen’s interest the most. And indeed, he was able to confirm that finding using brain samples from an independent cohort of patients.
Given all these hints, Andersen wondered if SorLA might be interacting with AβPP, which gets processed as it passes through the Golgi. To test this, Andersen used an affinity trap method to show that the extracellular domains of the three major forms of AβPP all bind to SorLA. With an analytical centrifuge he was able to demonstrate that the purified sorting protein forms a 1:1 complex with AβPP. When he used AβPP- or SorLA-specific antibodies to immunoprecipitate complexes from cells overexpressing the proteins, he found that they also bind to each other. Collaborating with Brad Hyman’s group at Massachusetts General Hospital in Charlestown, Andersen used fluorescence lifetime imaging to show that the two molecules lie adjacent to one another in N2a cells. The fluorescence lifetime of a fluorophore tagged to SorLA was significantly reduced when AβPP tagged with an acceptor fluorophore was also expressed in the cells, a sign that the donor and acceptor are in closer proximity than would be expected by pure chance. This experiment suggests that the interaction may be physiologically relevant.
Fluorescent staining then showed that both AβPP and SorLA co-localize in the perinuclear region of cells. In fact, when Andersen expressed a SorLA mutant that is missing the cytoplasmic domain and so ends up getting shunted rapidly to the cell surface instead of being localized to the Golgi, he found that SorLA and AβPP then co-localize on the cell surface. The finding suggests that SorLA might influence the cellular location of AβPP. In fact, when Andersen looked at the localization of AβPP in Chinese hamster ovary (CHO) cells, which do not express SorLA, he found that the precursor protein is predominantly in the endoplasmic reticulum, but when SorLA is expressed in CHO cells, the distribution of AβPP shifts to the Golgi and endosomes.
So what might the AβPP- SorLA interaction be doing? The obvious question is whether SorLA influences the proteolytic processing that leads to the shedding of the soluble AβPP ectodomains and prepares the precursor for the γ-secretase cleavage that generates Aβ. To get at this question, Andersen used SY5Y neuroblastoma cells, which produce endogenous AβPP. When SorLA was expressed in these cells, the levels of total AβPP were unaffected, but the shedding of both α- and β-secretase cleaved ectodomains (sAβPPα and sAβPPβ) declined dramatically, suggesting that SorLA protects against AβPP processing.
Recently, Andersen and colleagues have made SorLA knockout mice which express normal levels of AβPP but have levels of Aβ1-40 and Aβ1-42 elevated by about 30 percent, which is in keeping with the proposed role of the sorting protein in AβPP processing.
Stefan Lichtenthaler, working with Christian Haass’ lab at the University of Munich, Germany, also weighed in on the sorting theme. Lichtenthaler reported how he had used a cell-based functional assay to look for novel proteins that may influence the shedding of AβPP ectodomains. The assay system is based on an alkaline phosphatase-AβPP chimera expressed in HEK 293 cells. A simple phosphatase assay detects shedding of the phosphatase reporter after either α- or β-secretase activity.
After putting 20,000 clones through his screen, Lichtenthaler separated candidates into two camps: those that increased shedding by 1.5- to 3.0-fold, and those that increased it more than threefold. Some familiar names appeared, including the proposed α-secretase ADAM10, and BACE1, one of two β-secretases. These hits served to validate the method. In the second camp, Lichtenthaler found one clone that increased shedding by over fourfold. The gene, 788B8, is found on chromosome 18 of the human genome but poorly characterized. Lichtenthaler found that when expressed in the HEK cells, it has little effect on ectodomain shedding from tumor necrosis factor receptor 2 or L-selectin, two proteins that are also processed by α-secretase. This suggests that the effect on AβPP shedding is not due to some general regulation of domain shedding but is probably a specific effect.
Lichtenthaler then realized that 788B8 was none other than sorting nexin 30 (SNX30), an uncharacterized member of a family of exactly 30 sorting proteins that contain phosphoinositol binding domains (PX domain) and are involved in protein transport from and to the endosomes. PX domain proteins are generally associated with the cell membrane through their ability to bind to phosphoinositides in the lipid bilayer.
Lichtenthaler found that SNX30 is ubiquitously expressed in all tissues, including brain, and he further determined that it is a phosphoprotein. Asking whether SNX30 affects both α- and β-secretase cleavage of AβPP, Lichtenthaler tested conditioned medium from HEK293 and COS7 cells expressing the precursor protein. When SNX30 was transiently expressed in these cell lines, he found that there was a strong increase in sAβPPα in the medium from both cell types. The secretion of sAβPPβ, however, was hardly changed, suggesting that the sorting nexin preferentially affects α cleavage.
How might the nexin achieve this specificity? One possibility is that it may affect the distribution of AβPP. There is accumulating evidence to suggest that α cleavage takes place on the surface of the cell, while β cleavage takes place in endosomes. Any perturbation of trafficking through the endosomes could significantly affect the ratio of α-to-β cleavage.
To test if SNX30 might stabilize AβPP at the cell surface where it may be more amenable to α-secretase, Lichtenthaler determined AβPP uptake in the presence or absence of the sorting nexin. He added an antibody to AβPP to cooled COS7 cells expressing the precursor protein, warmed the cells up, and measured the time course of AβPP antibody uptake. In the presence of SNX30, the amount of endocytosed AβPP did increase with time, but only half as quickly as seen in cells not expressing the nexin, suggesting that SNX30 can affect endocytosis of AβPP. Lichtenthaler even speculated that the phosphorylation of SNX30 might regulate the distribution of the sorting nexin between the cytosolic and membrane fractions and thus serve as a switch that could control the uptake of AβPP.
Shedding of a protein can only occur if there is some kind of “sheddase” around. In the case of AβPP, such enzymes include the two β-secretases BACE1 and BACE2 and perhaps ADAM10, one of several metalloproteinases proposed to be the α-secretases (see ARF related news story and ARF related Sorrento coverage ). Some ADAM family members are also capable of self-shedding, chopping off their own ectodomains. If that is not complicated enough, Dieter Hartmann from Katholieke University Leuven, Belgium, reported yet another, incestuous twist to ADAM10 biology: The protease may be processed by none other than γ-secretase.
Hartmann’s group made this discovery following an investigation into ADAM10 shedding. Western blots carried out in his lab revealed a small, 8 kDa protein that cross-reacted with an ADAM10 antibody, giving the first indication that the ectodomain of this sheddase may itself be shed. In subsequent analysis, Hartmann’s group found that an N-terminal fragment of ADAM10 appears in the supernatant of cells expressing the protein.
Which protein might be responsible for releasing the ectodomain of ADAM10? To answer this question, Hartmann turned to a set of ADAM knockout cell lines. He found that only in ADAM9 knockouts was ADAM10 shedding severely curtailed, while in ADAM9/ADAM15 knockouts it was abolished, suggesting that ADAM9 and ADAM15 cooperate to cleave ADAM10.
Then what happens to ADAM10 after its ectodomain is shed? Could the C-terminal fragment (CTF) undergo further processing to yield an intracellular domain, much like AβPP does? If so, what purpose might that serve? Hartmann tested if presenilins, the major intramembrane proteases and catalytic component of γ-secretase, have any effect on ADAM10 processing. He showed that in presenilin-1 knockout cell lines, the ADAM10 CTF increased, suggesting that γ-secretase indeed plays a role. This ADAM10 CTF increase went down with expression of human presenilin-1 but not an inactive mutant form of the protease, and γ-secretase inhibitors reduced ADAM10 CTF processing. Finally, Hartmann detected the release into the cytoplasm of an internal C-terminal domain (ICD) of ADAM10.
It is unclear to what extent ectodomain shedding and the subsequent processing of ADAM10 is physiologically important. ADAM10 fragments do appear in different tissues including the liver, kidney, and brain, suggesting that the processing is not merely an artifact of cell culture, Hartmann said. But that is about as much as is known to date.
Likewise, scientists have not sorted out what purpose the release of the ICD might serve. Is it a recycling mechanism or might it be for signal transduction? Clues supporting a signaling role include the presence of a nuclear localization signal on the ICD of ADAM10, whereas most members of the ADAM family do not have one. Furthermore, when Hartmann expressed both ADAM9 and ADAM10 in COS cells, substantial amounts of the ADAM10 ICD ended up in the nucleus.
But perhaps the most intriguing aspect of this work is the finding that the soluble, shed ectodomain of ADAM10 retains proteolytic activity, can digest proteins on other cells, and can even digest Aβ peptides that retain the α-secretase cleavage site. ADAMs surely are getting curiouser and curiouser.—Tom Fagan.
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Sorrento: ADNI Imagines the Future of AD Imaging
With the myriad of different techniques that are now available to visualize changes in the brain, neuroimaging seems poised to make a real difference for people with neurodegenerative diseases. But at this point, it is well-nigh impossible to predict which of the multifarious incarnations of PET, MRI, and SPECT will win out and become standard practice for diagnosing, and monitoring the treatment of, Alzheimer disease and its harbinger, mild cognitive impairment (MCI). Add to that a second layer of confusion that arises from differences between imaging centers in the way they acquire and analyze data for what should be comparable approaches. This state of affairs, where many labs have developed their own approaches, calls for a broad effort to coordinate, validate, and hopefully reach consensus on the best ones, which scientists can then present to regulatory agencies in unison. Otherwise there is a danger that a weaker technology might be adopted for the wrong reasons.
At the 7th International Conference on AD/PD held earlier this month in Sorrento, Italy, Michael Weiner of the University of California at San Francisco outlined a new strategy that hopes to provide a clearer picture of the pros and cons of neuroimaging. Weiner is the principal investigator of the Alzheimer’s Disease Neuroimaging Initiative (ADNI). This five-year multi-site study aims to set standards for image acquisition, build a data repository, and determine which methods provide the most power when it comes to interpreting the efficacy of treatments in clinical trials.
The project is funded to the tune of $60 million. A third of that comes from industry, the remainder from the National Institutes of Health via the National Institute on Aging and the National Institute of Biomedical Imaging and Bioengineering. ADNI plans to spend the first six months establishing standard MRI and PET imaging techniques across the 40 to 50 sites throughout the U.S. and Canada that are expected to participate in the initiative.
This standard methodology will then be used to acquire images from the 800 study participants the project hopes to enroll by July 2006, reported Weiner. Four hundred will have MCI, 200 will have AD, and 200 will be control cases. ADNI will take images every six months, on average, for two to three years. At every time point, these images will be taken: 1.5 Tesla MRI images of all patients, FDG PET images of 50 percent, and 3-Tesla MRI images of 25 percent of participants. At every time point, participants will donate blood and urine samples. The project hopes that at least 20 percent of the volunteers will agree to give CSF samples at 0 and 12 months. Indeed, Weiner emphasized how precious these samples are and said he hoped that many more than 20 percent will agree to a spinal tap. Patients will also undergo a clinical assessment at each visit.
The project is ambitious and will be quite arduous for the patients, said Weiner. One of the major difficulties he foresees is getting volunteers to agree to participate in this lengthy study knowing that they will not receive any non-standard treatment. ADNI hopes that by emphasizing the importance of the study for future patients, they will be able to recruit enough participants to meet their enrollment goals.
Over the five years, ADNI will establish a brain imaging and biomarker database, and develop improved methods for monitoring trials. Tasks will be divided between a clinical and a neuroimaging core. Weiner stressed that all data will be freely and rapidly available to anyone who wants to view them online. Some of the specific aims of the project are:
- to develop imaging standards akin to the ADAS-COG scores that are currently used to measure cognition;
- to improve methodology;
- to determine optimum methods for acquiring and processing images and biomarkers;
- to validate the imaging and biomarker data by correlating it with neuropsychiatric behavioral data;
- to provide a database and biological samples for the pharmaceutical industry.
Other investigators are: Leon Thal at University of California, San Diego, who heads the Alzheimer’s Disease Cooperative Study (ADCS) core; Clifford Jack, head of the MRI core, and Ron Peterson at the Mayo Clinic, Rochester, New York; William Jagust at the University of California, Berkeley, head of the FDG-PET core; John Trojanowski at the University of Pennsylvania, Philadelphia, head of the biomarker core (see his recent magazine article on the promise of biomarkers and how ADNI will help in their discovery); Arthur Toga at the University of California, Los Angeles, head of the informatics core; Laurel Beckett at the University of California, Davis, head of the statistics core. Peter Sneider from Pfizer serves as chair of the industry advisory board.
The imaging data will be available at the Laboratory of Neuro Imaging (LONI) at UCLA and can be freely downloaded for analysis. A clinical database run by Leon Thal’s group at the Alzheimer’s Disease Cooperative Study will also be freely accessible. Anticipating that distribution of biomarker samples could become a contentious issue, ADNI has enlisted the help of an independent, outside advisory committee that will be set up by the NIA and chaired by Flint Beal at Cornell University, New York. This committee will take requests for biomarker samples and decide how to divvy up the specimens. For more information on ADNI, go to the project’s website.—Tom Fagan.
No Available Further Reading
Sorrento: Trouble with the Pro’s
No, this is not a story, published in the wrong place, about a misbehaving sports star. It is about protein growth factors in Alzheimer disease. Apparently, the pro-form of nerve growth factor is an obnoxious player in early AD, whereas its mature form tries to play defense. This is the upshot from the 7th International AD/PD meeting in Sorrento, Italy, where several speakers tried to clarify exactly what role growth factors play in the devastating loss of cholinergic neurons that characterizes AD. Researchers know that cholinergic neurons depend for their survival on both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and both have received considerable attention of late as potential therapeutics for Alzheimer and other neurodegenerative diseases. NGF has made it to a phase I gene therapy clinical trial for AD (see ARF related news story). But more recently, the growth factor waters became muddied when scientists realized that NGF in its precursor form, proNGF, can actually promote cell death rather than survival.
This means that the fate of cholinergic neurons may depend on a delicate balance between NGF, proNGF, and their respective receptors, tyrosine receptor kinase A (TrkA) and the neurotrophin receptor p75NTR. In fact, Margaret Fahnestock at McMaster University, Hamilton, Canada, recently showed that there is very little NGF in the human brain, whereas proNGF is easily detectable and gradually increases as patients progress from no cognitive impairment (NCI) to mild cognitive impairment (MCI) and on to mild and severe AD (see Peng et al., 2004). In addition, many groups, including Elliott Mufson’s at Rush University Medical Center, Chicago, have demonstrated that levels of tyrosine receptor kinase A (TrkA), the “pro survival” receptor for NGF, are reduced in both the nucleus basalis and cortex of people with mild to severe AD (see Hock et al., 1998, Counts et al., 2004, and Mufson et al., 1997), and that levels of TrkA correlate quite well with cognitive function.
These findings suggest that the loss of the pro-survival TrkA receptor, coupled with the increase in the pro-apoptotic proNGF could create an environment that contributes to the disappearance of cholinergic neurons with AD progression.
In Sorrento, Mufson gave an update on his ongoing analysis of samples taken through the Religious Order Study on aging and Alzheimer disease. This study conducts yearly cognitive and neurological examinations of hundreds of catholic priests, nuns, and brothers at over 40 religious communities throughout the U.S. who have all agreed to donate postmortem tissue. It has already provided invaluable information on cognitive decline (see, for example, ARF related news story on the link between AD and Parkinson disease), or more recently Bennett et al., 2005).
In his latest examination of postmortem samples, Mufson focused on the deterioration of basal forebrain cholinergic neurons and their cortical projections, and the relationship between that deterioration and NGF/proNGF signaling. Last October, Mufson and colleagues reported that in contrast to TrkA, cortical levels of p75NTR, which activates a neuronal cell death pathway, were unchanged in people with mild cognitive impairment (MCI) or mild to severe AD (see Counts et al., 2004). In Sorrento, he reported that in the cholinergic basal forebrain (CBF), neurons staining for both p75NTR and TrkA are reduced in both MCI and mild AD compared to NCI, whereas numbers of neurons expressing choline acetyltransferase (ChAT) are similar among the three groups. This suggests that dysfunctional neurotrophin signaling may precede the loss of cholinergic transmission in the CBF, said Mufson.
Corroborating this protein and immunocytochemistry data were the results from expression analyses on custom microarrays of single cholinergic neurons aspirated from tissue samples. This work is a collaboration with Steven Ginsberg at the Nathan Kline Institute in New York. Mufson reported that there are no significant differences between gene expression of p75NTR in MCI, early AD, or NCI, but levels of TrkA transcripts did turn out to be down in samples from MCI patients. Expression of ChAT was no different among the three groups, again pointing to neurotrophin dysfunction as a forerunner of cholinergic transmission problems. The loss of p75NTR in the basal forebrain neurons, despite the apparent normal expression of the protein, suggests that perhaps degradation of the receptor is accelerated in this particular region of the brain. In addition, the fact that p75NTR levels are maintained in cortical projection sites despite the phenotypic reduction in the expression of p75NTR in neurons of the basal forebrain indicates that other cells may be compensating by increasing expression of p75NTR in the cortex, suggested Mufson. All told, the data indicate that there may be a progressive loss of the “good” NGF signaling while the “bad” proNGF signals are maintained as patients progress.
Further support for the “too-little-NGF, too-much-proNGF” hypothesis comes from Antonio Cattaneo at Lay Line Genomics, Rome, and the International School for Advanced Studies, Trieste, Italy. Cattaneo has developed a line of transgenic mice, the AD11 mice, which are deficient in basal forebrain NGF because they express a high-affinity anti-NGF antibody in the central nervous system (see ARF related news story). These mice exhibit AD-like symptoms including long-term potentiation and behavioral deficits, and cortical degeneration. In addition, they have elevated levels of Aβ1-40 and Aβ1-42, plaque pathology, and tau pathology.
How much of this pathology might go back to changes in the cell survival versus cell death pathways activated by mature versus proNGF? Cattaneo reported that in the AD11 mice, the anti-NGF antibody reacts poorly with proNGF in the first place, and when it does bind it dissociates faster than it does from the mature trophin. Perhaps the antibody is neutralizing the mature form of NGF while leaving the pro-apoptotic immature form free to act through the p75NTR receptor and its recently identified binding partner sortilin? (See Nykjaer et al., 2004 and ARF related Sorrento news on the relationship of two other sorting proteins, SorLA and sorting nexin 30 to Aβ precursor protein processing).
To test this idea, Cattaneo bred AD11 mice with p75NTR knockout animals. Because these animals are missing the extracellular domain of p75NTR, signaling through the receptor is lost while signaling through the TrkA receptor should still work. These AD12 crosses have a less severe phenotype than their AD11 parents, Cattaneo revealed. Though cholinergic neurons begin to die in AD12 mice at two months—as they do in AD11 mice—by six months the cholinergic deficit has been completely rescued in AD12 animals. Amyloid deposits are also much reduced: At six months, amyloid deposition is down 20-fold compared to AD11 mice and at 15 months, AD12 animals have almost no amyloid deposition.
The tau pattern is not so simple, however. At two months, AD11 mice show little hyperphosphorylated tau (P-tau), but AD12 mice contain a large number of P-tau neurons in both the cortex and the hippocampus, reported Cattaneo. He has not yet worked out how to explain this. Even so, he suggested that the two forms of NGF can have profound consequences for the well-being of neurons depending on how their processing is modulated, and how their availability and signaling changes.
Margaret Fahnestock also addressed the role of mature versus immature trophic factors, in the progression of AD, but focused on brain-derived growth factor (BDNF). Researchers have known for a decade that BDNF mRNA levels are down in the hippocampus (see Murray et al., 1994). That study, using just a handful of postmortem samples from AD patients, has stood up well as numerous others have confirmed and extended it, including a report from Fahnestock’s group showing that levels of proBDNF are also reduced, by up to 40 percent, in the parietal cortex of AD patients (see Michalski and Fahnestock, 2003).
But are all BDNF transcripts to be tarred with the same brush? As Fahnestock pointed out, humans produce at least seven different BDNF transcripts and different promoters regulate their expression. This raises the possibility that there may be differential expression of the BDNF family members in response to the challenges posed in the AD brain.
To address this, Fahnestock used reverse-transcriptase PCR to measure levels of specific transcripts in human brain tissue. She reported that transcripts 1, 2, and 3 are all downregulated in the AD brain compared to controls. In keeping with this, levels of proBDNF were reduced in samples from the Religious Order Study by about a third in both MCI and AD patients, compared to NCI.
This downregulation might be caused by none other than Aβ. Fahnestock reported that when she treated SY5Y neuroblastoma cells with Aβ1-42, it downregulated expression of the BDNF 3 transcript and led to a reduction in the total amount of BDNF in the cells. In contrast to NGF, it appears that people with MCI and early AD lose both the mature and immature forms of BDNF, and that Aβ may play at least a partial role in reducing transcription of the trophin. This suggests yet another avenue whereby the amyloid peptide might compromise neuronal function.—Tom Fagan.
- Orlando: Preliminary Results of NGF Gene Therapy Trial Are Mixed
- Tau and α-synuclein at the Nexus of Alzheimer's and Parkinson's
- NGF, Galantamine Rescue Anti-NGF Model of Alzheimer's
- Sorrento: Sorting Out Shedding of Ectodomains
- Peng S, Wuu J, Mufson EJ, Fahnestock M. Increased proNGF levels in subjects with mild cognitive impairment and mild Alzheimer disease. J Neuropathol Exp Neurol. 2004 Jun;63(6):641-9. PubMed.
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- Counts SE, Nadeem M, Wuu J, Ginsberg SD, Saragovi HU, Mufson EJ. Reduction of cortical TrkA but not p75(NTR) protein in early-stage Alzheimer's disease. Ann Neurol. 2004 Oct;56(4):520-31. PubMed.
- Mufson EJ, Lavine N, Jaffar S, Kordower JH, Quirion R, Saragovi HU. Reduction in p140-TrkA receptor protein within the nucleus basalis and cortex in Alzheimer's disease. Exp Neurol. 1997 Jul;146(1):91-103. PubMed.
- Bennett DA, Schneider JA, Bienias JL, Evans DA, Wilson RS. Mild cognitive impairment is related to Alzheimer disease pathology and cerebral infarctions. Neurology. 2005 Mar 8;64(5):834-41. PubMed.
- Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow TE, Hempstead BL, Petersen CM. Sortilin is essential for proNGF-induced neuronal cell death. Nature. 2004 Feb 26;427(6977):843-8. PubMed.
- Murray KD, Gall CM, Jones EG, Isackson PJ. Differential regulation of brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase messenger RNA expression in Alzheimer's disease. Neuroscience. 1994 May;60(1):37-48. PubMed.
- Michalski B, Fahnestock M. Pro-brain-derived neurotrophic factor is decreased in parietal cortex in Alzheimer's disease. Brain Res Mol Brain Res. 2003 Mar 17;111(1-2):148-54. PubMed.
No Available Further Reading
Sorrento: Tumor Suppressor Ties Miscreant Kinase to Cell Cycle
Exactly what makes p25/Cdk5 so deadly to neurons? A good place to start tracing the path of this rogue kinase would be retinoblastoma (Rb), a tumor suppressor protein positioned strategically at the crossroads between cell proliferation and death. This is the upshot of a poster presented at the 7th International AD/PD Conference earlier this month in Sorrento, Italy. Luc Buée and colleagues at INSERM in Lille, France, with collaborators at Aventis Pharma, reported that inducing p25 in neurons prompted an inactivating phosphorylation of the Rb protein, which then allowed the transcription of genes pushing otherwise differentiated cells to start dividing.
During the past decade, researchers in a growing number of labs, most prominently Inez Vincent’s, have implicated an aberrant reactivation of the cell cycle in the pathogenesis of Alzheimer disease, claiming that this process contributes early on to the neuronal loss seen in AD (see, for example, ARF related news story and ARF news story). One of the many questions surrounding this hypothesis is what happens upstream, i.e., what forces quiescent neurons to reenter the cell cycle?
Independently, other laboratories including Li-Huei Tsai’s have accused a pathogenic cleavage product of the physiological Cdk5 regulator p35 of playing a role in AD, ALS, and stroke. These investigators believe that noxious stimuli, ranging from Aβ and elevated calcium to H2O2 or glutamate, can activate the protease calpain, which then produces p25. Whereas scientists consider p35/Cdk5 a tightly controlled, physiological, membrane-tethered kinase, they view p25/Cdk5 as a hyperactive maniac engaged in a phosphorylating spree throughout the cytoplasm and the nucleus (see ARF related news story and ARF news story).
It’s established that p25/Cdk5 can trigger neurodegeneration, but how it does so is less clear. Scientists have focused on two possible explanations: phosphorylation and dysregulation of the microbutule binding protein tau, or something untoward happening with the cell cycle. Intrigued by a Live Discussion on the Alzheimer Research Forum, Buée, himself long a student of tau, decided to pursue the question of the cell cycle.
His group first established a stable neuronal cell line that expresses an inducible p25/Cdk5 activity (Hamdane et al., 2003). In Sorrento, he presented evidence that, as early as six hours after p25/Cdk5 induction, this kinase phosphorylates Rb. P-Rb then activates the transcription factor E2F and its downstream genes, some of which are cell cycle genes documented to be aberrantly active in AD. The kinases that normally phosphorylate Rb were not active in this system. Roscovitine, an inhibitor of Cdk5 but not most other cyclin-dependent kinases, abolished the p25/Cdk5-induced change in Rb. Finally, p25/Cdk5 isolated from the cells directly phosphorylated Rb in vitro, the researchers report.
The work suggests that Rb phosphorylation is an early event in p25/Cdk5-induced neurodegeneration, Buée said (see Hamdane et al., 2005). More broadly, this would imply that Cdk5 deregulation sets off an inappropriate phosphorylation of several different substrates, including Rb, placing Cdk5 upstream of cell cycle changes in the pathogenesis of AD.
The French scientists performed their latest work in differentiated neuroblastoma cells. Usually, inducing p25 in such cells causes apoptosis. To circumvent this problem, first author Malika Hamdane simultaneously overexpressed tau, deploying it as a phosphorylation reservoir to absorb some of the rampant p25 activity. This allowed the cells to survive long enough for Hamdane to detect the Rb phosphorylation and subsequent activation of cell cycle genes.
Buée readily acknowledges that pathway hypotheses generated from experiments with cultured cell lines and overexpressed genes all share the caveat that the cells used may well activate different signal transduction pathways than do aging human neurons. Therefore, Buée is planning to check if his lab’s finding holds up in vivo, for example, in p25-inducible mouse strains.—Gabrielle Strobel.
- AD Cell Cycle Reentry—Early Rather Than Late
- Linking APP with Cell Cycle Reentry and Apoptosis—One Kinase Does the Trick
- Orlando: It’s Getting Hot around CDK5, Has the Field Noticed Yet?
- The Calpain Connection
- Hamdane M, Sambo AV, Delobel P, Bégard S, Violleau A, Delacourte A, Bertrand P, Benavides J, Buée L. Mitotic-like tau phosphorylation by p25-Cdk5 kinase complex. J Biol Chem. 2003 Sep 5;278(36):34026-34. PubMed.
- Hamdane M, Bretteville A, Sambo AV, Schindowski K, Bégard S, Delacourte A, Bertrand P, Buée L. p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death. J Cell Sci. 2005 Mar 15;118(Pt 6):1291-8. PubMed.
- Hisanaga S, Saito T. The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator. Neurosignals. 2003 Sep-Oct;12(4-5):221-9. PubMed.
- Arendt T. Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity. Prog Neurobiol. 2003 Oct;71(2-3):83-248. PubMed.
- Monaco EA, Vallano ML. Role of protein kinases in neurodegenerative disease: cyclin-dependent kinases in Alzheimer's disease. Front Biosci. 2005 Jan 1;10:143-59. PubMed.
- Hoozemans JJ, Veerhuis R, Rozemuller AJ, Arendt T, Eikelenboom P. Neuronal COX-2 expression and phosphorylation of pRb precede p38 MAPK activation and neurofibrillary changes in AD temporal cortex. Neurobiol Dis. 2004 Apr;15(3):492-9. PubMed.
- Raina AK, Zhu X, Smith MA. Alzheimer's disease and the cell cycle. Acta Neurobiol Exp (Wars). 2004;64(1):107-12. PubMed.
- Guo Q. Cyclin-dependent kinase 5--a neuronal killer?. Sci Aging Knowledge Environ. 2003 Dec 17;2003(50):pe36. PubMed.
- Zhang M, Li J, Chakrabarty P, Bu B, Vincent I. Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Niemann-Pick Type C mice. Am J Pathol. 2004 Sep;165(3):843-53. PubMed.
Sorrento: More Mice and Men—What Role ApoE in Vascular Dementia?
When most people think of Alzheimer disease (AD), they imagine parenchymal amyloid plaques. But vascular deposits can be just as damaging. Consider familial dementias caused by the Dutch (E693Q) or Iowa (D694N) mutations in AβPP (see review on Aβ mutations and their physiological effects). Underlying their cognitive and memory defects, these patients have extensive vascular deposits of Aβ, inflammation in and around the vessels, and they are prone to small hemorrhages (see related ARF Live Discussion). One pressing question in the field is how ApoE, the leading risk factor for AD, affects vascular Aβ deposits.
In Sorrento, Bill Van Nostrand from Stony Brook University, New York, addressed this issue when he reported how apolipoprotein E affects amyloid deposits in a mouse model of cerebral amyloid angiopathy (CAA). Previously, he had shown that Tg-SwDI transgenic mice, which express human AβPP harboring Dutch, Iowa, and Swedish (K670N/M671L) mutations, develop early onset deposits of Aβ in both the brain parenchyma and the cerebral vasculature. The parenchymal deposits are diffuse, while deposits in the blood vessels are fibrillar. In-vivo transport studies have shown that these mice have trouble clearing Aβ across the blood-brain barrier (see Davis et al., 2004), though mice with only the Swedish mutation—identical in every other respect to the Tg-SwDI mice—can clear Aβ across the blood-brain barrier, supporting the notion that the Dutch and Iowa mutations somehow affect clearance of Aβ from the brain.
To evaluate what effect ApoE might have on plaque pathology in Tg-SwDI animals, Van Nostrand and colleagues crossed their mice with animals lacking ApoE. The resulting Tg-SwDI/ApoE-negative mice express human AβPP at normal levels, and produce total levels of Aβ1-40 and Aβ1-42 on par with those in Tg-SwDI parents. However, Van Nostrand reported that the microvascular deposition of Aβ, as judged by both immunostaining and thioflavin S reactivity, was almost completely eliminated in animals lacking ApoE, while parenchymal deposits were reduced by about 50 percent. (The only exception was the subiculum, part of the hippocampus, where Aβ deposits increased by about twofold.) The reductions, it turns out, may be due to an effect on Aβ fibrillization. That’s because though the total levels of Aβ are the same in these animals, insoluble Aβ levels were reduced by about 50 percent, and soluble Aβ levels were concomitantly doubled. In fact, an ELISA revealed a 50 percent reduction in oligomeric forms of Aβ in the insoluble fraction. Antibodies developed by Bill Klein at Northwestern University, Chicago (see ARF related news story), which are specific for soluble oligomers, revealed that these species are increased about twofold in the soluble fraction, Van Nostrand reported.
Mice in the ApoE-negative background also had no inflammation surrounding the blood vessels, Van Nostrand reported. Stereological measurements revealed highly significant decreases in the numbers of activated microglia and reactive astrocytes surrounding the microvessels of the cortex and thalamus. This might explain why the animals performed better, Van Nostrand added. On the rotorod test, Tg-SwDI mice do poorly compared to controls (scores are about 50 percent lower), but in the ApoE-/- background their performance was back to normal. (Tg-SwDI mice also exhibit impaired learning and memory in the radial water maze test; ApoE-negative animals are currently being evaluated.)
Work from David Holtzman’s lab, Washington University, St. Louis, has also implicated ApoE in Aβ clearance and deposition in cerebral vessels (see Fryer et al., 2005), and he presented some of that data at Sorrento. Holtzman has found that mouse and human ApoE affect Aβ deposition in the mouse brain very differently. While his data on ApoE-/- mice is in general agreement with results from Van Nostrand’s lab, in mice expressing human ApoE the story is different. Human ApoE delays the onset of Aβ deposition in mice. And when these animals do eventually develop deposits, the ones expressing the ApoE4 isoform get them first, followed by ApoE3, then ApoE2 animals. What’s more, in mice expressing human ApoE4, the burden of the deposits is shifted from the parenchyma to the blood vessels. Holtzman has found that this may be due to an increase in the Aβ40:Aβ42 ratio. The smaller Aβ1-40 peptide has been shown to be more prone to deposition in the vasculature (see ARF related news story), and in mice expressing human ApoE4, levels of Aβ1-40 are higher early in life.
Exactly how mouse ApoE accelerates vascular deposition of Aβ is not well understood. Mouse and human ApoE are obviously not behaving in the same way. Van Nostrand believes that removal of mouse ApoE shifts the Aβ balance in favor of more soluble forms of the peptide. In the absence of adequate clearance mechanisms, these would then accumulate in the brain.
Another way for ApoE to influence brain Aβ would be to alter clearance. It is worth noting that Holtzman has shown that mouse ApoE, in cooperation with ApoJ, aids clearance (see ARF related news story and ApoE symposium). One thing is clear: By figuring out how and why human and mouse ApoE’s differ, much could be learned about Aβ production, deposition, and elimination.—Tom Fagan.
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- Davis J, Xu F, Deane R, Romanov G, Previti ML, Zeigler K, Zlokovic BV, Van Nostrand WE. Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor. J Biol Chem. 2004 May 7;279(19):20296-306. Epub 2004 Feb 25 PubMed.
- Fryer JD, Simmons K, Parsadanian M, Bales KR, Paul SM, Sullivan PM, Holtzman DM. Human apolipoprotein E4 alters the amyloid-beta 40:42 ratio and promotes the formation of cerebral amyloid angiopathy in an amyloid precursor protein transgenic model. J Neurosci. 2005 Mar 16;25(11):2803-10. PubMed.
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