DC: Funding We Can Believe In? Perhaps, But Scientists Must Advocate

21 Nov 2008

On November 15, as representatives of the world’s 20 richest nations were meeting in Washington, DC, to try to hash out ways to stem global economic hemorrhaging, more than 31,000 scientists began arriving a few blocks away for the 38th annual meeting of the Society for Neuroscience (SfN). The coincidence did not go unnoticed. For many neuroscientists, and junior faculty in particular, the top priority after neurons is finding the support to study them. Over the coming days, the Alzforum will report on some of the scientific studies presented at this year’s conference, but we start with a story that is of interest to not only all Alzheimer’s researchers, but all neuroscientists: how will the recent election and current economic slowdown affect your chances of getting funded?

That question was the subject of an SfN Public Advocacy Forum, organized well before Election Day by John Morrison, Mount Sinai School of Medicine, New York, and held on November 18. At the forum, entitled “The Elections: And the Winner Is...Science?,” there was palpable optimism that when President-elect Barack Obama is sworn in on January 20, the current dire funding situation will change for the better. (The NIH budget has not seen a real increase since 2003, and at present manages a meager 10 percent success rate for grants.) This not-so-audacious hope is nourished by what panelist Katrina Kelner, Deputy Editor of Life Sciences at Science Magazine, called the “post-election glow.” It was tempered by a dose of fiscal realism from the three other panelists: former NIH Director Harold Varmus, now president and CEO of Memorial Sloan-Kettering Cancer Center, New York; Wendell Primus, Senior Policy Advisor to Speaker of the U.S. House of Representatives, Nancy Pelosi; and the Honorable John Porter, Chair Research!America Board of Directors.

Despite the sobering economic climate, the take-home message from this discussion was that an increase in science funding might well be realized. But there was a catch—scientists must take an active role in lobbying lawmakers. Quoting Obama, Morrison, who chairs the SfN Government and Public Affairs Committee, said, “Everyone needs to roll up their sleeves and get involved in finding solutions.” The panel echoed this sentiment.

Varmus said that the current situation is not unlike that when President Clinton appointed him director of the NIH in 1993. Grant approval success rates had dropped precipitously—partly because the length of grant had been extended—and there were not only obstacles to increasing the budget but also suggestions of a 5 percent cut to all agencies. Instead, Congress enacted a bill that doubled the NIH budget over five years starting in 1998. Since 2003, NIH funding declined by 14 percent in real terms, which Porter, a former Republican representative of Illinois, called a “disaster.” During that time, he said, discretionary federal spending went to defense, homeland security, and veterans’ affairs. Everything else was flat funded. That, said Porter, highlights the challenge ahead. The desire to consider other priorities—education, labor, national parks, for example—is very strong. “This was the most exciting election in my lifetime. People are hugely inspired and have had their expectations raised,” said Porter, but he added that on January 20, 2009, those expectations will come home to roost.

Primus agreed. He said that though the $100 billion stimulus package currently being considered includes $1 billion for the NIH, its chance of passage in the lame duck session is slim. Primus predicted that lawmakers will muddle on through December, and major funding issues will fall to Obama on January 20. When President Bush was inaugurated in 2001, the Congressional Budget Office estimated a $5.6 trillion national surplus, while on January 20 President Obama will inherit a $7 to $9 trillion deficit, Primus said. “Funding for you depends on reducing that deficit, and that depends on taxes,” said Primus. “You have to make the case that investments have long-term payoff.”

Porter believes that payoff is a robust economy. He suggested that if the country is not devoted to science, innovation, and research, then the economy will stagnate. Without continued investment in basic and translational research, the U.S. will have trouble competing with the rest of the world. Varmus suggested also arguing for short-term value, that funding science pays for salaries for researchers and non-researchers alike, and for purchase of supplies and the support that goes with it. Funding science acts as its own stimulus package, Varmus said.

The panelists emphasized that to boost funding, scientists need to engage not only members of Congress but also the public. Scientists are a respected segment of the community, suggested Porter, and urged the audience to use that to their advantage. “Write a letter to the editor, an op-ed piece, get people into your lab or office and talk science to them,” he suggested.

For his part, Morrison listed four steps scientists can take:

Sign up for the SfN Advocacy Network
Take part in Capitol Hill Day—meet members of Congress and their aides
Conduct a lab tour for elected representatives
Become a science advisor to your local representative

Porter advised that in talking to lawmakers, scientists should have a concise plan for what they want the lawmakers to do because after listening for 20 minutes, that is what they will ask for. Varmus and Porter both argued for aiming for a predictable, reasonable increase in funding. Porter suggested 3 percent a year adjusted for inflationary costs in the medical research field.

What is likely to happen on the funding front after January? Primus predicted that the first bill to be passed may be an economic recovery bill, which will deal with unemployment benefits, food assistance, and assistance to states. The second bill will likely be an appropriations bill, which may increase funding for the NIH, CDC, and other agencies. After that, Primus expects carryovers from last year to come up, including the Health Information Technology Bill and the FDA Tobacco Bill. Primus argued for increased funding for clinical research, specifically comparative drug trials, which could eventually help reduce prescription drug costs. This is likely to be controversial.

As for the executive branch, though Varmus chaired Obama’s science advisory committee, he said that stint was over and that he does not have the ear of the President-elect. Porter noted that how quickly the new president will name a science advisor to the Office of Science and Technology Policy, and whether this will be a cabinet-level position will be early indicators to the priority of scientific research in the new administration. After that, Obama’s first State of the Union address will lay out the agenda for years ahead, Porter said.—Tom Fagan.

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DC: Aβ Clearance—Roles for MBP, Transcription Factors?

26 Nov 2008

Gumming up the brain years before a person may notice memory loss or other hints of Alzheimer disease, amyloid-β (Aβ) has fueled much of AD research, and efforts to develop Aβ-targeted therapeutics have consumed billions of dollars. With more than 200 talks and posters devoted to some aspect of the pesky peptide, this year’s Society for Neuroscience (SfN) meeting, held 15-19 November in Washington, DC, left our small crew of Alzforum reporters with considerable ground to cover. In the coming days, we will highlight a small handful of recent Aβ developments, focusing primarily on unpublished data and work not previously covered on the Alzforum. This article describes two new studies on Aβ clearance—one proposing myelin basic protein (MBP) as a novel Aβ-degrading enzyme, another presenting serum response factor (SRF) and myocardin (MYOCD) as transcriptional co-activators that regulate clearance of brain amyloid. Aβ metabolism is under increased scrutiny in part because accumulating evidence suggests that Aβ immunotherapy works well to decrease plaque load, but may need help from other clearance mechanisms to completely flush Aβ out of the brain vasculature (ARF related news story). As always, ARF welcomes your comments on these and/or other SfN presentations on related topics.

MBP may be best known for its involvement in multiple sclerosis. Indeed, antibodies to the protein, a major component of the myelin sheaths that insulate nerve fibers, play a role in MS pathogenesis. Still, the new SfN data isn’t the first to link MBP to AD. Several studies have suggested that neurodegeneration in AD tracks with myelination patterns during growth and maturation (Bartzokis et al., 2006; Bartzokis et al., 2004). Another investigation identified myelination of peripheral neurons as a major physiological function of BACE1, which catalyzes the first of two proteolytic steps needed to release Aβ from its precursor (see Willem et al., 2006 and ARF related news story). Last year, William Van Nostrand and colleagues at Stony Brook University in New York reported that MBP could bind mutant Aβ that causes cerebral amyloid angiopathy (CAA) and inhibit its assembly into fibrils in vitro (Hoos et al., 2007). Curiously, they noticed in those assays that if MBP was incubated with Aβ for longer periods, the Aβ seemed to disappear. Consistent with this observation, another group reported autocatalytic activity in MBP purified from human brain, and suggested that this cleavage mechanism could play a role in generating autoreactive peptides in MS (D’Souza et al., 2005). Meichen Liao, a graduate student in Van Nostrand’s lab, reasoned that if MBP has enzymatic activity, this behavior could explain the vanishing Aβ seen in the earlier in vitro studies. Based on her emerging data presented publicly for the first time at SfN, the answer appears to be “yes.”

Assessing degradation of monomeric Aβ by purified human MBP in vitro, Liao found that after 24 hours, Aβ40 levels dropped by 50 percent and Aβ42 dipped to about 20 percent of starting levels. In a cell culture—Cos-1 cells transfected with MBP—levels of exogenous Aβ40 and Aβ42 fell by 50 and 60 percent, respectively, after 48 hours. To address whether MBP can degrade fibrillar Aβ, Liao and colleagues performed two in vitro assays. In the first, they coaxed synthetic Aβ42 peptide to form fibrils (by incubating at 37 degrees C for six days), then used thioflavin T fluorescence to determine the change in Aβ fibril content over two days in the presence or absence of MBP. For the second assay, the team mixed unlabeled and FITC-labeled Aβ42 in a 9:1 ratio, aged them for six days at 37 degrees C to promote fibril formation, then two days later, assessed fibrillar Aβ content as the fluorescence of pelleted material in samples treated with or without MBP. By both assays, the amount of fibrillar Aβ dropped by about 50 percent after MBP treatment compared to controls.

Having established that MBP degrades monomeric and fibrillar Aβ in vitro, Liao tested whether MBP could degrade Aβ deposits formed in vivo in Tg2576 mice, a widely studied AD model that overexpresses mutant human amyloid precursor protein (APP) and develops both plaques and vascular Aβ deposits. When fresh cortical slices from the brains of aged Tg2576 mice were incubated with purified human MBP, both plaque load and vascular amyloid, detected as thioflavin S staining, dropped about 60 percent relative to pre-MBP levels, Liao reported. This effect was blocked by addition of the serine proteinase inhibitor, phenylmethane sulfonylfluoride (PMSF).

“These are still early days for this particular study, but I think they've got some nice data showing there's something interesting going on,” said Cindy Lemere of Brigham and Women's Hospital and Harvard Medical School, Boston, in a phone conversation after the SfN meeting.

During Q&A and in conversations after the talk, several scientists expressed concern that the experiments could have picked up enzymatic activity of a contaminant in the purified human MBP preps. Liao said that silver stain gels and mass spectrometry experiments have consistently identified a single protein in their MBP samples. Furthermore, MBP purified from a variety of cell types—including human white matter, mouse brain, bacterial recombinant systems, and adenovirus-infected mammalian cells—seems to exhibit the same activity, she said, reducing the likelihood of a contaminating enzyme in the preps. To Lemere, the experiments showing Aβ degradation in transfected Cos-1 cells, which do not normally express MBP, provided the most compelling evidence against the contamination possibility. “That would be pretty remarkable if the same factor that's in the brain extracts is in the Cos-1 cells,” she told ARF.

Speaking with this reporter at the SfN meeting, Van Nostrand acknowledged limitations with interpreting the data thus far. “Right now we’re just looking at purified MBP,” he said. “We don’t know if it has this effect when wrapped around myelin sheaths.” To address whether native MBP can influence Aβ accumulation in vivo, his group is crossing MBP knockout mice with two APP-overexpressing transgenic models—Tg2576 and another strain, SwDI (Davis et al., 2004), which expresses a human APP gene with Swedish, Dutch, and Iowa mutations, leading to a massive buildup of vascular amyloid and parenchymal plaques in the brain.

During the same SfN session, Robert Bell, a graduate student in the lab of Berislav Zlokovic at the University of Rochester, New York, described a transcriptional mechanism that may regulate clearance of vascular Aβ in the brain. This work, which will appear in the February issue of Nature Cell Biology (DOI: 10.1038/ncb1819), extends an earlier study by Zlokovic's group showing that AD patients have increased levels of the transcriptional co-activators myocardin (MYOCD) and serum response factor (SRF) (see ARF related news story). MYOCD is restricted to cardiac muscle and smooth muscle cells, whereas SRF is found in all cell types. Bell and colleagues found that MYOCD/SRF pathway regulates Aβ clearance via low-density lipoprotein receptor-related protein 1 (LRP1), a key Aβ clearance receptor (see ARF related news story).—Esther Landhuis.

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References

News Citations

Paper Citations

Bartzokis G, Lu PH, Geschwind DH, Edwards N, Mintz J, Cummings JL. Apolipoprotein E genotype and age-related myelin breakdown in healthy individuals: implications for cognitive decline and dementia. Arch Gen Psychiatry. 2006 Jan;63(1):63-72. PubMed.
Bartzokis G, Sultzer D, Lu PH, Nuechterlein KH, Mintz J, Cummings JL. Heterogeneous age-related breakdown of white matter structural integrity: implications for cortical "disconnection" in aging and Alzheimer's disease. Neurobiol Aging. 2004 Aug;25(7):843-51. PubMed.
Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, Destrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006 Oct 27;314(5799):664-6. PubMed.
Hoos MD, Ahmed M, Smith SO, Van Nostrand WE. Inhibition of familial cerebral amyloid angiopathy mutant amyloid beta-protein fibril assembly by myelin basic protein. J Biol Chem. 2007 Mar 30;282(13):9952-61. PubMed.
D'Souza CA, Wood DD, She YM, Moscarello MA. Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry. Biochemistry. 2005 Sep 27;44(38):12905-13. PubMed.
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.

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DC: Primate, Mouse Studies Sustain Aβ Immunotherapy Hopes

26 Nov 2008

Loaded with promise and disappointment, the roller-coaster tale of Alzheimer disease vaccine development has raised hopes and dampened some, but mostly leaves researchers and the AD community at large ever watchful. At the Society for Neuroscience (SfN) annual meeting, held 15-19 November in Washington, DC, several preclinical studies hinted that efforts to refine AD immunotherapy approaches seem to be on the right track. While arousing new questions, the data might also bolster an underlying hunch that given enough time and tweaks, Aβ-targeted immunotherapy should succeed. In its first test in primates, a dendrimeric N-terminal Aβ vaccine reduced plaque burden and improved cognition in aging Caribbean vervets, scientists reported in Washington, DC. Meanwhile, in mice that recapitulate human disease more fully than many earlier AD transgenic models, active immunization with full-length Aβ42 not only reduced amyloid and tau pathology but also prevented neuron loss and reversed memory deficits.

In 2002, Phase 2 trials of the first AD vaccine to reach human testing—Elan/Wyeth’s AN1792—came to a halt when treated participants developed encephalitis (see ARF related news story). Subsequent investigation (see, e.g., Pride et al., 2008) traced the harmful inflammation to Aβ-specific T cells produced in response to immunization. Attempting to sidestep these adverse events, Elan/Wyeth came out with a new AD vaccine using a seven-amino acid immunogenic sequence from the Aβ N-terminal but lacking the C-terminal epitope believed to trigger Aβ-specific T cell activation (see Agadjanyan et al., 2005). Called ACC-001, clinical testing of this vaccine also stumbled in Phase 2 when a patient developed vasculitis, or inflammation of the blood vessels. This serious adverse event forced Elan and Wyeth to temporarily suspend dosing in their trial earlier this spring (see ARF related news story), though the trial resumed and is again recruiting patients (see ARF news story).

Other drug candidates that look promising in mice have met similar ill fates when they entered human testing. To bridge this gap, some scientists are testing their therapies in primates, though most of these preclinical studies are small and unpublished. Several years ago, Cindy Lemere of Brigham and Women's Hospital and Harvard Medical School and colleagues reported that 10 months of active immunization with full-length Aβ reduced CNS amyloid deposition in four aged vervets with early AD pathology, compared to 10 age-matched controls (Lemere et al., 2004). While this pilot study demonstrated that vervets are a suitable AD model with amyloid levels that could be lowered by active Aβ immunization, the researchers could not at the time address whether the vaccine actually improved cognition.

At the SfN meeting, Lemere presented data from a more recent Elan/Wyeth-funded study involving 22 vervets aged 18-24 years. Lemere noted that at the mean age in this study (20.5 years), vervets show some plaque deposition, though most don't have severe AD pathology. At this age, their amyloid pathology roughly corresponds to the early stages of AD, Lemere told the audience. The vervets were randomized into three groups using age, gender, CSF Aβ levels, and behavioral performance as selection criteria. The control group received QS-21, an adjuvant used in the AN1792 formulation, without Aβ. The treatment groups received QS-21 with either full-length Aβ42 or dendrimeric Aβ1-15 (dAβ1-15). With 16 copies of Aβ1-15 peptide linked on a branched lysine core—a molecular trick for beefing up short peptides to drive a strong immune response—dAβ1-15 lacks the C-terminal epitope thought to induce self-reactive, Aβ-specific T cells. In earlier mouse studies, Lemere and colleagues showed that this vaccine triggers a robust antibody response and lowers plaque burden in APP transgenic mice immunized intranasally (Seabrook et al., 2006). T cells activated by this vaccine did not recognize endogenous full-length Aβ, suggesting that this approach might avoid the harmful inflammation that plagued human trials of earlier AD vaccine candidates.

In the study presented at SfN, each group of vervets received seven intramuscular injections over 9.5 months. All animals immunized with full-length Aβ produced whopping specific antibody titers, detected by ELISA using Aβ40-coated plates. In the dendrimeric vaccine group, most animals made Aβ-specific antibodies at titers that were reasonable though lower than those seen in animals that received full-length Aβ, Lemere said. All antibody responders showed reduced plaque burden.

Importantly, these amyloid reductions appeared to do some good. By the end of the study, vervets immunized with full-length Aβ kept up their performance on two cognitive measures, whereas non-vaccinated animals had deteriorated. Vervets in the dendrimeric Aβ group actually showed cognitive improvement on both tests, and it was significant, Lemere said in her talk. She added that her group was surprised as to how such strong cognitive gains could come with specific antibody titers that were lower than those in the full-length Aβ group. The Aβ40 coated ELISA might have missed certain conformation-specific Aβ epitopes, or the dendrimeric vaccine might have induced a different kind of immune response. The team will next address the apparent discrepancy between the cognitive effect and the strength of specific immune response.

While the work of Lemere’s group suggests that N-terminal Aβ vaccines may be a way to go, data from another preclinical study presented at the SfN meeting show that full-length Aβ works well in APP-overexpressing mice that model human disease better than do most other AD rodent lines. Despite their significant amyloid and sometimes tau pathology, many Alzheimer’s mouse models lack a defining feature of AD—significant neuronal loss. By knocking out the gene for nitric oxide synthase 2 (NOS2), Donna Wilcock, Carol Colton, and Mike Vitek at Duke University Medical Center in Durham, North Carolina, enabled the primarily vascular amyloid deposition in Tg-SwDI mice (Davis et al., 2004) to expand to tau pathology and neuronal loss (see ARF related news story).

The researchers tested how active Aβ immunotherapy would fare in these mice (APPSwDI/NOS2-/-) and in another strain they had generated earlier (APPSw/NOS2-/-), which has less severe, predominantly parenchymal Aβ pathology (see ARF related news story). They immunized year-old mice with fibrillar Aβ1-42 or a keyhole limpet hemocyanin (KLH) carrier control, both containing Freund’s adjuvant. At the start of vaccinations, both strains of mice showed extensive amyloid and tau pathology as well as hippocampal neuron loss and cognitive deficits, representing early to mid-stage AD. By the end of the study at 16 months, vaccinated APPSwDI/NOS2-/- mice showed a 30 percent drop in brain Aβ and 30-40 percent reduced tau hyperphosphorylation. The Aβ immunizations also slowed (but did not halt) neuron loss and partially rescued spatial memory deficits, as measured by a radial arm water maze. Effects in the APPSw/NOS2-/- group were more dramatic. Vaccinated animals had a 65-85 percent reduction in brain Aβ and 50-60 percent reduced levels of hyperphosphorylated tau. Remarkably, the vaccine prevented any further neuron loss and completely reversed memory deficits in these mice.

The only real downer in this study was the presence of microhemorrhages in many of the vaccinated APPSw/NOS2-/- mice. The APPSwDI/NOS2-/- mouse, curiously, does not bleed, normally or with vaccination, Wilcock told ARF. “We hypothesize that is because the CAA in this mouse is primarily capillary in nature, so it is possible the vessel becomes occluded by amyloid prior to the occurrence of leakage,” she said. “This is purely speculative at this point.” She noted that CAA in AD is mostly in smaller arteries and arterioles, as is CAA in APPSw/NOS2-/- mice, which might explain why those animals showed microhemorrhages in the recent study. Though an obvious safety concern, it is unclear if microhemorrhages carry functional ramifications, and studying them is challenging because of their varying extent and localization. “When you’re dealing with seven or eight mice, and each one has (microhemorrhage) in a different place, you really can’t pull out any consequences of that,” Wilcock told this reporter. As reported recently by Dora Games and colleagues at Elan Pharmaceuticals, South San Francisco, California, research in PDAPP mice seems to indicate that microhemorrhage can be mitigated by lowering the dose of N-terminal Aβ antibodies offered for six months as passive immunotherapy (Schroeter et al., 2008 and ARF related news story).

Whether this translates to human studies remains unclear, but a study published last month by James Nicoll of the University of Southampton, U.K., and colleagues supports the idea that Aβ immunization sucks Aβ out of plaques, leading to a transient increase in cerebral amyloid angiopathy (CAA) severity as the Aβ gets drained out of the brain through the perivascular system (Boche et al., 2008). In that investigation of AD patients immunized against Aβ42, Nicoll’s team found that those who survived until four to five years after the initial vaccination had virtually no remaining plaques or CAA, raising the possibility that Aβ is cleared from the cerebral vasculature with time. Games told ARF that her group has unpublished data hinting that a similar clearance may happen in PDAPP mice receiving passive Aβ immunotherapy. “We've done experiments out to nine months, and it looks to us that the incidence of these small microhemorrhages decreases over time,” she said.

Interestingly, even after intense scrutiny of nine brain areas, Lemere’s team did not see microhemorrhage in any of the vervets in their most recent study. When asked about these lesions, Lemere attributed their routine occurrence in mouse studies to the start of vaccinations comparatively later in the disease process, when animals already have severe pathology. “If your animal model has a lot of CAA or vascular amyloid at the time of immunization, whether active or passive, if all of a sudden you've got a buildup of antibodies in the periphery, it makes it more likely you're going to run the risk of having microhemorrhage,” Lemere said. “That's one reason I'm a proponent of vaccinating early, even pre-symptomatically. Not only are younger people better at generating an antibody response, but most younger people don't have amyloid deposition in their blood vessels.”—Esther Landhuis.

References

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Paper Citations

Pride M, Seubert P, Grundman M, Hagen M, Eldridge J, Black RS. Progress in the active immunotherapeutic approach to Alzheimer's disease: clinical investigations into AN1792-associated meningoencephalitis. Neurodegener Dis. 2008;5(3-4):194-6. 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.
Seabrook TJ, Thomas K, Jiang L, Bloom J, Spooner E, Maier M, Bitan G, Lemere CA. Dendrimeric Abeta1-15 is an effective immunogen in wildtype and APP-tg mice. Neurobiol Aging. 2007 Jun;28(6):813-23. PubMed.
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.
Schroeter S, Khan K, Barbour R, Doan M, Chen M, Guido T, Gill D, Basi G, Schenk D, Seubert P, Games D. Immunotherapy reduces vascular amyloid-beta in PDAPP mice. J Neurosci. 2008 Jul 2;28(27):6787-93. PubMed.
Boche D, Zotova E, Weller RO, Love S, Neal JW, Pickering RM, Wilkinson D, Holmes C, Nicoll JA. Consequence of Abeta immunization on the vasculature of human Alzheimer's disease brain. Brain. 2008 Dec;131(Pt 12):3299-310. PubMed.

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DC: New Neprilysin Methods Reduce Brain Aβ

26 Nov 2008

Neprilysin, one of a handful of proteases known to degrade amyloid-β (Aβ), might seem an attractive therapeutic for Alzheimer disease (AD)—until you consider the difficulties of administration. Direct injection into the brain has shown some success in animal models, though it is hardly ideal for treating a chronic neurodegenerative disease in humans. But perhaps there’s another way. At the Society for Neuroscience annual meeting in Washington, DC, held 15-19 November, researchers described means of boosting neprilysin activity and reducing amyloid load in transgenic mouse models of the disease. They ranged from increasing neprilysin activity in the blood to protein- and cell-based methods of delivering it into the periphery and then getting it across the blood-brain barrier. The latter method, which uses monocytes to ship neprilysin to the vicinity of plaques, had been proposed as a general approach before, but this is the first time anyone has shown that it works, according to Dave Morgan, University of South Florida, Tampa. The monocyte approach has potential beyond AD.

In a poster presentation, Yinxing Liu, from the laboratory of Lou Hersh at the University of Kentucky, Lexington, reported that boosting neprilysin in the circulation of transgenic mice can reduce Aβ deposits in the brain. Liu used a retroviral approach to inject a neprilysin-expressing construct into the hind leg of nine-month-old 3xTG mice (Oddo et al., 2003). The secreted form of mouse neprilysin reached levels of 400-1,000 nmol product/min/ml as measured in an assay using an artificial substrate (activity in normal plasma is ~0.2 nmol/min/ml). After three months of this, the plasma levels of Aβ dropped from about 7 pM to 4.5 pM. Interestingly, brain Aβ also fell. This might be explained by the peripheral sink hypothesis, which suggests that lowering Aβ in the blood eventually pulls it out of the brain as well. This has been seen with other therapeutic approaches (see ARF related news story), including vaccines (see ARF related news story). Liu reported that in addition to Aβ deposits being cut in half, soluble Aβ in the brain was down by almost a third compared to untreated transgenic controls, but he reported no changes in behavior.

Researchers at Eliezer Masliah’s lab at the University of California, San Diego, also described a viral-neprilysin approach. In a slide talk, Brian Spencer reported a lentiviral construct that targets neprilysin for passage across the blood-brain barrier (BBB). The construct fuses a secreted form of neprilysin with the low-density lipoprotein receptor-binding domain of apolipoprotein B. In theory, this domain should help ferry the neprilysin in and out of cells and into the brain.

Spencer reported that even though it is conjugated to the ApoB domain, the secreted neprilysin degrades Aβ in vitro. In vivo, three months after a single intraperitoneal injection of the vector into transgenic mice expressing APPSwe under the Thy 1 promoter, the fusion protein was present in the brain and brain neprilysin activity went up. The increase was accompanied by a reduction in Aβ deposits and soluble Aβ monomers. APP and oligomeric Aβ levels were not changed. Spencer said he was not sure why oligomers were unaffected, but that other neprilysin studies had reported the same thing. “There may not be a direct line from the monomeric Aβ to oligomeric Aβ to plaques,” Spencer told ARF via e-mail. Rather, the plaques might be one outcome of accumulation of monomeric Aβ and free oligomers may be another outcome. “Further studies need to be performed to determine the exact relationship neprilysin plays in the accumulation of other Aβ species,” he said.

Spencer had no behavioral data to show whether this approach improves learning and memory in the transgenic animals, but he did show that the construct was found predominantly near neurons and glia in the dentate gyrus of the hippocampus.

“One of the problems with current methods of delivering neprilysin is that they are all intracranial, which would not be feasible for humans,” Spencer said. Dave Morgan of the University of South Florida, Tampa, agrees. “At best, using intracranial injection, we can get neprilysin into about one third of the mouse brain,” Morgan commented. “The human brain is 1,000 times the volume of the mouse brain, which means we’d have to turn it into a pin cushion if we wanted to use intracranial delivery,” he said. Instead, Morgan’s lab has come up with a novel way of sneaking neprilysin into the brain—by expressing it in monocytes. If practical in humans, this approach would be used beyond AD. “Any CNS disorder that has a significant macrophage activation component could be theoretically amenable to treatment using this technique,” Morgan said.

In her poster presentation, Lori Lebson from Morgan’s lab demonstrated how monocytes expressing a secreted form of neprilysin prevent buildup of Aβ plaques in transgenic mice. Lebson isolated GFP-expressing macrophages from a mouse line and transfected them with a plasmid that expresses a secreted form of neprilysin (the membrane-binding domain is replaced with a secretory signal). First Lebson injected these neprilysin-secreting monocytes directly into the cortex and hippocampus of 15-month-old double transgenic mice (APP/PS1) to determine if they would do any good. She found a drop in soluble Aβ and in Congo red-positive deposits after one week, whereas a control experiment using an inactive neprilysin construct did not.

Next Lebson tested if monocytes injected into the blood could enter the brain and degrade Aβ. Several labs have shown that circulating immune cells can cross the BBB in AD mouse models (see ARF related news story). Morgan noted that it has been theorized that monocyte therapy could work, though no one has been able to prove it. Lebson injected five million monocytes twice weekly into nine-month-old transgenic mice via a microvascular port attached into the jugular vein; this gives a more consistent injection pattern than trying to inject into mouse tail veins, for example, and allows for repeated injections over weeks. After two months, the researchers found that the monocytes completely reduced the buildup of new Aβ plaques but that it had no effect on plaques that were already in the brain. “That’s a very important point,” said Morgan. “Very few people are measuring Aβ load at initiation, but without that data point we would be saying we reduced amyloid load by half.” Over the two months, plaque load doubled in untreated mice.

Intriguingly, while the researchers found that monocytes entered the brain of transgenic mice and congregated in the vicinity of plaques, they found that absolutely no monocytes found their way into the brain of control animals after injection. This indicates that there has to be some signal from the brain, or perhaps damage to the BBB, which facilitates the entry of monocytes into the brain of transgenic models.

Morgan said the advantage of this method is that the therapy can be directly targeted to the site where it is needed. In addition, it could be used to deliver other genes or treat other diseases. “We are not so interested in neprilysin as in showing that the monocyte therapy itself can work,” he said. In the end, neprilysin may not be the best therapeutic approach because as a protease it is fairly “promiscuous,” he said, and so may have untoward effects.

Morgan suggested that the way forward using this methodology is first to show that it would work in an acute setting, where a patient’s own blood cells are transfected and re-introduced. Because monocytes are short-lived, this approach would be relatively safe. In the mouse circulation, for example, the GFP monocytes were undetectable within 90 minutes and lasted only about a week in the brain. If that holds true in humans as well, then treatment could be withdrawn easily. For a long-term therapy, Morgan predicted that a viral approach would have to be used to transfect patients’ own stem cells to keep an Aβ-targeting monocyte population going over the long run. Monocytes are known to change phenotype when they enter the brain. Therefore, the transfected, therapeutic gene should be driven by those promoters that get activated when the cells make this phenotypic switch, suggested Morgan.—Tom Fagan.

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DC: Dogs May Provide First Natural Animal Model for ALS

29 Nov 2008

The mSOD1 mouse, harboring a mutated human superoxide dismutase (SOD1) gene, has long held the spotlight as the model of choice for amyotrophic lateral sclerosis (ALS). It has yielded scores of papers on disease progression and potential treatments, but it is far from a perfect model. At the Society for Neuroscience annual meeting, held 15-19 November in Washington, DC, veterinary researchers from the University of Missouri in Columbia reported that canine degenerative myelopathy, an inherited condition with ALS-like symptoms, is linked to a mutation in the canine superoxide dismutase gene, as are 20 percent of familial ALS cases. Neurons from afflicted dogs showed SOD1 aggregates reminiscent of ALS pathology. Study authors Joan Coates and Dennis O’Brien of the University of Missouri in Columbia, along with other collaborators at the University of Missouri and at the Broad Institute of MIT/Harvard, suggested that the dogs could serve as a valuable model for ALS.

There is only one drug for ALS, and no treatment exists for canine degenerative myelopathy (DM). “We want to help the dogs and also help people,” O’Brien said. Dogs have served as valuable testing grounds for cancer therapies, the authors noted, and these animals might serve the same purpose in testing ALS treatments. The researchers first described the link between DM and SOD at the 19th International Symposium on ALS/MND, held 3-5 November in Birmingham, U.K.

Canine degenerative myelopathy and ALS share the late onset of a progressive motor neuron disease and the SOD1 mutation and pathology. The main difference between the conditions is that while the vast majority of human SOD1 mutations associated with ALS are dominant, the dog mutation is recessive, apparently with incomplete penetrance. Dogs have a shorter lifespan than people; disease onset is nine to 11 years and the canine disease progression is faster than in humans, from between six and 18 months depending on breed. “This means that the results of any treatment can be assessed much faster in dogs than in people,” wrote William Thomas, a veterinary neurologist at the University of Tennessee in Knoxville, in an e-mail to ARF. Thomas was not involved with the study.

Mutant SOD1 mice, in contrast to the dogs, exhibit an artificial disease. Scientists engineered the animals to express unnaturally high levels of mutant human SOD1. “In these mice…SOD1 has 10-fold the activity as normal,” Coates said. “You may not see the true clinical picture there.” The mice are convenient models because they reliably develop disease and progress to end-stage within months. However, the mSOD1 mouse has been criticized because several treatments that appeared promising in mice failed to benefit humans (Schnabel, 2008). “This [dog] model may more closely represent the clinical situation in the human population,” Coates said.

“I think it’s a neat potential tool, but there’s a lot more that has to be done,” said Jeffrey Rothstein, a neurologist at Johns Hopkins University in Baltimore, Maryland, whose research includes ALS therapeutics. Scientists have not yet defined the details of DM progression in dogs, and a deep clinical understanding of the disease is necessary to produce significant results in a therapy trial. Even in genetically identical mice, Rothstein said, it took a decade to work out the disease progression needed to make the animals experimentally useful.

Degenerative myelopathy, or DM, was first recognized in German shepherds in the 1970s, and was long believed to be specific to the breed (Averill, 1973). It was also thought to only affect the spinal cord. Because owners often choose to euthanize a pet once it becomes partially paralyzed, it was not until recently that Coates and others were able to characterize the later stages of disease, particularly in Pembroke Welsh Corgis, another breed that suffers from DM. Being much smaller than German shepherds, Corgis are more easily nursed through the later stages of disease, providing researchers with further data on progression. Scientists found that in later-stage animals, motor neurons were affected as well. Sclerosis occurs in the lateral and dorsal spinal cord funiculi, peripheral nerves have axonal loss, and muscles atrophy. Similarly, the degeneration of motor neurons and axonal loss in ALS causes muscle atrophy. “The disease progression and clinical spectrum are similar to upper motor neuron ALS,” Coates said. Boxers, Rhodesian ridgebacks, and Chesapeake Bay retrievers are also susceptible to DM.

To trace the genetic cause of DM, Coates and O’Brien collaborated with Gary Johnson, also at the University of Missouri, and Kersten Lindblad-Toh and Claire Wade at the Broad Institute in Cambridge, Massachusetts. With support of the American Kennel Club-Canine Health Foundation, they carried out genomewide association mapping. This pointed to a potential DM locus on canine chromosome 31, which includes the dog SOD1 gene. “There were some excited phone calls about that,” O’Brien said. Resequencing of SOD1 revealed a missense mutation that changes canine SOD1 glutamate 40 to a lysine. Of 100 dogs with DM, 96 were homozygous for the SOD1 E40K mutation, suggesting the allele is recessive. (The other four animals, which were homozygous wild-type, likely had a different condition that mimicked DM symptoms, Coates said.) A codon for amino acid 40 of human SOD1 lies within a cluster of missense mutations that are associated with human ALS, including a glutamate-to-glycine point mutation at the position homologous to the canine E40K mutation.

Among healthy animals from the five breeds known to be susceptible to DM, there was an even distribution of wild-type homozygotes, mutant homozygotes, and heterozygotes. This suggests the allele is common in those five breeds, and that it shows incomplete penetrance. However, since many of the homozygous mutant animals were young, it is possible they might develop the disease later.

Mutant human SOD1 forms aggregates, and in neurons from ALS patients the protein is found in Lewy body like hyaline inclusions. To look for similar pathology, collaborator Martin Katz of the University of Missouri stained lower spinal cord sections from 19 dogs, covering all three genotypes, with rabbit anti-SOD1 antibody. In contrast to the diffuse SOD1 staining in neurons from normal dogs, all animals with DM had small, punctate SOD1-positive inclusions in their neurons. The heterozygote neurons exhibited an intermediate phenotype, inclusions that lightly stained for SOD1. While the dog SOD1 inclusions were small, not exactly like the large aggregates found in humans, the authors noted that the dogs were euthanized and may not represent end-stage disease as postmortem human tissues do.

The potential value of DM as a model for ALS lies in the quicker progression of the disease and the less stringent regulations for testing veterinary therapeutics. It would probably not be practical, O’Brien said, to breed mSOD1 dogs for research purposes. Since the age of onset is nine to 11 years, the costs for such a colony would be prohibitive. However, Coates thinks there are enough dogs within the general population to make a study sample; the overall disease prevalence of DM among all dogs is 0.19 percent, according to The Veterinary Medical Database, and the prevalence in certain breeds is likely to be much higher.

“This research represents a big advance in the understanding of degenerative myelopathy,” Thomas wrote “It provides useful clues regarding the underlying cause, it allows the first diagnostic test for the disease, and over the long term, it should help breeders plan their breeding strategy to hopefully decrease the incidence of this disease.” However, the prevalence of the mSOD1 allele means that breeders may not be able to completely eliminate it from the gene pool.

It’s not certain if DM is becoming more common or simply more commonly diagnosed. Purebred animals are particularly vulnerable to inherited disorders, and top show performers can cause a “popular sire effect” when bred extensively, leading to an increase in disease prevalence. “I really think over the years it has become more of a problem,” Coates said. Early symptoms are spastic paraparesis and ataxia; owners often notice their dogs stumbling and dragging their nails on the ground. The dogs also lose general proprioception in their limbs. If the researchers turn the animal's paw over, dogs with DM are not as quick to return it to normal position as a healthy dog.

The study authors are working on further pathological analysis of disease, such as examining cell body loss in the ventral horn and associated nerve loss, and developing tools to assess uniformity of disease progression, such as a rating scale for gait and electrophysiologic techniques. Since purebred animals are more alike genetically than people, it’s possible that the disease progression will be fairly uniform within each breed. If not, Rothstein said, “You need a lot of animals to get an answer. If you don't have stable numbers, you can never do therapeutics.” The exact value of dogs with DM as an ALS model, then, has yet to be determined.—Amber Dance.

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DC: More MicroRNA Implicated in Dementia

29 Nov 2008

Once thought to be a simple go-between, RNA is recently attracting much more attention as an important regulator of gene expression in its own right. At the Society for Neuroscience annual meeting, 15-19 November in Washington, DC, Rosa Rademakers of the Mayo Clinic in Jacksonville, Florida, presented evidence for the involvement of a microRNA (miRNA) in frontotemporal dementia, which is the cause of 10 to 20 percent of early onset dementia cases. The research is published in the December 1 Human Molecular Genetics.

MicroRNAs can degrade or repress translation of their targets, and also upregulate mRNA expression in some cases. The short sequences, 21 to 23 nucleotides, have been linked to neurodegenerative diseases such as Alzheimer’s (see ARF related news story and ARF news story); for review, see Bushati and Cohen, 2008), and Rademakers predicts that miRNA dysfunction will be implicated in many more conditions.

Rademakers’ research is the first evidence for miRNA involvement in frontotemporal dementia. She and colleagues found that the human miRNA miR-659 binds to the 3’-untranslated region of the progranulin mRNA, blocking translation. People with a progranulin mutation that enhances this binding can have significantly lowered progranulin levels, leading to frontotemporal dementia, Rademakers said, making the mutation a major risk factor for disease.

Scientists had previously linked frontotemporal lobe degeneration with ubiquitin- and TDP-43-positive inclusions (FTLD-U) to mutations in the progranulin gene (PGRN) (Baker et al., 2006, Cruts et al., 2006, and see ARF related news story). (Another form of frontotemporal dementia exhibits tau-positive inclusions.) All of the more than 60 PGRN mutations previously associated with FTLD-U are dominant, loss-of-function mutations that cause premature termination of the PGRN mRNA (Gass et al., 2006). These mutations cut progranulin protein levels by approximately 50 percent in heterozygotes, which is sufficient to cause disease (Van Damme et al., 2008).

Progranulin’s role in the nervous system is unclear. It acts as an anti-inflammatory in its full-length form but, when proteolytically cleaved to form granulins, is pro-inflammatory. Progranulin is present in neurons and microglia. In the periphery, progranulin is involved in wound healing (for review, see He and Bateman, 2003).

Rademakers and colleagues discovered the link between miRNA-659 and progranulin when they sequenced PGRN genes from 378 FTLD patients. Among 339 patients with no PGRN coding mutation, they found a polymorphism, rs5848, in the 3’UTR that did not exhibit Hardy-Weinberg equilibrium: more of the patients (55) were TT homozygous at this position than would be predicted by chance. TT homozygotes made up 16.2 percent of the FTLD patients, compared to 9.3 percent of control subjects. Among 59 FTLD-U patients without other FTLD-linked mutations, the rs5848 TT genotype frequency was 25.4 percent. All TT subjects showed the same neuropathology, with neuronal cytoplasmic inclusions and short, thin neurites in the cortex. The similar pathology suggests a common cause of disease among these patients. While the coding-sequence PGRN mutations are dominant, heterozygotes for rs5848 TT did not have increased susceptibility to FTLD-U, suggesting the mutation is recessive.

The location of the mutation, outside the coding sequence, suggests that it affects PGRN expression rather than function. On Western blots, extracts from rs4858 TT patients had progranulin levels reduced by approximately 30 percent compared to CC homozygous control extracts. However, quantitative RT-PCR found no difference in the PGRN mRNA levels between CC and TT individuals. “That was the first time we thought maybe microRNAs are involved,” Rademakers said. Using computer analysis, Rademakers predicted that a single miRNA, miR-659, could bind to the site of the mutation. In silico, changing the cytosine to a thymine altered the PGRN mRNA-miRNA binding, shifting miR-659’s position and allowing it to bind the transcript more tightly, with three additional nucleotide pairings. After confirming that miR-659 is expressed in human brain tissue, including the frontal and temporal neocortex, Rademakers hypothesized that miR-659 binds to PGRN mRNA, blocking translation, and that the mutant mRNA binds miR-659 more tightly. This knockdown of PGRN, in turn, could cause FTLD.

To test this hypothesis, the scientists turned to cell experiments. When they transfected human M17 neuroblastoma cells with miR-659, they expressed less progranulin, while cells transfected with a nonspecific control miRNA had normal PGRN levels. To further quantify miR-659’s activity, Rademakers and colleagues engineered a luciferase reporter with the PGRN 3’-UTR. In mouse N2A neuroblastoma cells (used to avoid any effects from endogenous human miR-659), miR-659 decreased luciferase expression in a dose-dependent manner, further suggesting miR-659 acts on the GRN 3’-UTR to knock down expression.

Rademakers posits that the lower one’s progranulin levels, the higher the risk of developing FTLD-U. A single T allele appears not to lower PGRN translation below the acceptable threshold, but two mutations can repress enough GRN translation to cause disease.

“The data do look convincing,” said Alison Goate, a geneticist at Washington University in St. Louis. “They provide pretty good evidence that there is an increased risk of FTLD-U associated with people who are homozygous for this variant.” However, she noted that the patient numbers were small—only 59 subjects in the second genetic analysis, and only 14 for the GRN expression studies. “In some ways, they were pretty lucky they found it,” Goate said.

MicroRNAs appear poised to be major players in neurodegenerative disorders, and Rademakers said her research is more evidence for that hypothesis. However, she recognizes that other scientists may take some convincing. “People are very skeptical of these novel mechanisms that become popular,” she said. Goate also noted that TDP-43 is implicated in several neurodegenerative diseases, and that perhaps GRN has a role in promoting TDP-43-based diseases other than FTLD-U. In the future, Rademakers intends to analyze sequence variation and expression levels of miRNAs to further characterize their role in FTLD.—Amber Dance

References

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Paper Citations

Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Berger Z, Eriksen J, Robinson T, Zehr C, Dickey CA, Crook R, McGowan E, Mann D, Boeve B, Feldman H, Hutton M. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature. 2006 Aug 24;442(7105):916-9. PubMed.
Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Vandenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, Kumar-Singh S, Van Broeckhoven C. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature. 2006 Aug 24;442(7105):920-4. PubMed.
Gass J, Cannon A, Mackenzie IR, Boeve B, Baker M, Adamson J, Crook R, Melquist S, Kuntz K, Petersen R, Josephs K, Pickering-Brown SM, Graff-Radford N, Uitti R, Dickson D, Wszolek Z, Gonzalez J, Beach TG, Bigio E, Johnson N, Weintraub S, Mesulam M, White CL, Woodruff B, Caselli R, Hsiung GY, Feldman H, Knopman D, Hutton M, Rademakers R. Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration. Hum Mol Genet. 2006 Oct 15;15(20):2988-3001. PubMed.
He Z, Bateman A. Progranulin (granulin-epithelin precursor, PC-cell-derived growth factor, acrogranin) mediates tissue repair and tumorigenesis. J Mol Med (Berl). 2003 Oct;81(10):600-12. PubMed.

DC: Amyloid-Laden Brains—What Do They Mean for Healthy Seniors?

05 Dec 2008

Widely seen as the molecular trigger for a cascade of neurological and behavioral changes leading to Alzheimer disease, Aβ lurks within the brains of many cognitively normal seniors, too. Does the presence of fibrillar amyloid in these folks foretell future dementia? Based on several studies presented at the Society for Neuroscience (SfN) annual meeting in Washington, DC, held 15-19 November, the answer hovers around a not-so-straightforward “probably.” Using a full arsenal of brain imaging technology including positron emission tomography (PET) and magnetic resonance imaging (MRI), the new investigations are pinpointing the neurological features and cognitive abilities associated with amyloid deposition, and may help determine whether these changes reflect normal aging or early signs of disease. Meanwhile, preliminary data from a study of “super agers” hints that staying mentally sharp in the golden years may depend less on Aβ and correlate more with the ability to stave off tau pathology.

In a slide talk, Elizabeth Mormino, a graduate student in the lab of William Jagust at the University of California, Berkeley, told the audience that live brain imaging using the PET radiotracer Pittsburgh Compound B (PIB) routinely detects amyloid in 10-40 percent of non-demented elderly. Comparing brain amyloid levels of normal controls from the Berkeley Aging Cohort with those found in an independent cohort of UC San Francisco AD patients, she said that “on the whole, amyloid levels are higher in the AD patients, but there is some overlap.” She and colleagues analyzed whether amyloid load correlated with reduced hippocampal volume and episodic memory in this group of 20 dementia-free Berkeley seniors, and in two other groups of non-demented elderly—17 normal controls and 39 PIB-positive mild cognitive impairment (MCI) patients—from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Mormino noted that only the PIB-positive MCI individuals were analyzed because PIB-negative MCI patients often convert to non-AD dementias, perhaps reflecting the presence of pathologies not related to amyloid.

Across all three cohorts, individuals with greater amyloid deposition had smaller hippocampi, Mormino reported. However, an association between higher PIB index and poorer episodic memory was less consistent—showing up in the Berkeley cohort and in the PIB-positive MCI ADNI group, but not in the ADNI normal control group. These observations led the researchers to suspect that “maybe these three variables are related but in a specific way,” Mormino said. “Maybe hippocampal volume mediates the relationship between PIB and episodic memory.” To test that idea, her team performed regression analysis looking at how PIB load contributes to episodic memory. When they corrected for hippocampal volume, PIB was no longer significantly associated with episodic memory, she reported. However, regardless of whether they accounted for PIB levels, hippocampal volume remained a significant predictor of episodic memory. Consistent with a model in which Aβ deposition, hippocampal atrophy, and episodic memory loss occur sequentially in nondemented elders, the findings suggest that the relationship between Aβ and episodic memory is indirect and possibly mediated by hippocampal damage.

During the same SfN slide session, Keith Johnson of Massachusetts General Hospital and Harvard Medical School in Boston presented PIB data hinting that amyloid in the brains of healthy older people may in fact spell impending doom. “What we attempted to do was to take a look at individuals who are normal, who have amyloid binding, and see after a follow-up period whether their neuropsychological function had changed,” Johnson said. In Washington, he expanded on findings described earlier this spring by Harvard Medical School colleague Dorene Rentz at the Human Amyloid Imaging conference in Chicago (see ARF related conference story). In the study, 31 non-demented older adults (15 with Clinical Dementia Rating [CDR] scores of 0, 16 with CDR 0.5) received PIB-PET scanning and cognitive tests at baseline, and follow-up cognitive assessment about a year later—test scores were adjusted for age, education, estimated IQ, and baseline cognitive performance. During the study’s short timeframe, greater amyloid deposition in the precuneus correlated with memory decline, most prominently in 30-minute delayed recall. “This is not a substantial, clinically apparent decline in memory function. It’s really memory performance in a very specific way that we’re detecting here,” he said, noting that tests of executive function, language function, and visuospatial function did not show this trend. Still, the results are intriguing given that memory decline of any sort showed up after just one year, and that the changes were related to amyloid buildup in the precuneus. “The precuneus is a very good proxy. It’s the region that leads the pack in terms of amyloid deposition,” said Johnson, adding that longitudinal follow-up is required to nail down whether brain amyloid in normal elderly predicts later progression to dementia.

Efforts to probe the functional significance of fibrillar brain amyloid in normal older adults were also described in an SfN poster by Trey Hedden of Massachusetts General Hospital. Collaborating with Johnson and others, Hedden used a combination of neuroimaging and neuropsychological techniques to address how Aβ pathology in otherwise healthy seniors relates to various measures of cognition and neural function that change with age. The researchers compared three groups of people: 36 healthy university students (ages 18-27), and a cohort of 29 healthy older adults (ages 61-84) subdivided into PIB+ (n = 17) and PIB- (n = 12) groups. As determined by diffusion tensor MRI, the elder participants differed from their younger counterparts in certain measures of white matter integrity that typically change with age, but these alterations were seen regardless of PIB status.

On the other hand, amyloid load did seem to matter for functional assessments involving the default network, a set of brain areas that fire up when the mind is resting and tone down during focused mental tasks. Among older participants, who as a group fared worse than the younger adults, PIB+ individuals underperformed their PIB- counterparts on several attentional control tasks. In addition, MRI measurements of correlated brain activity (activity measured during a task but that has been temporally filtered to remove task effects), revealed age-related disruptions that were worsened by the presence of amyloid. Previous work has shown that AD patients and memory-impaired older adults have reduced default activity (see ARF related news story and ARF news story), and it may be that specific parts of this network are differentially affected by aging and AD. Interestingly, PIB-dependent differences did not show up in the attention-task MRI data. Hedden speculates that the correlated connectivity measurements could be “a more sensitive measure of aging or disease-related effects because they represent the spontaneous functional coherence of the brain.” He suggested that when faced with specific tasks, cognitively normal older adults might be able to recruit compensatory reserves to confront possible difficulties, which could mask differences that would appear in the unfocused, spontaneous state. The bottom line, suggested Harvard Medical School colleague and coauthor Reisa Sperling in an e-mail to ARF, is that “the presence of amyloid does disrupt normal function in the default mode network, similar to the disruption reported in early AD. Thus, PIB imaging may be particularly useful in better defining the process of normal versus pathologic aging.”

Other researchers are tackling this question from a different angle. Instead of studying what goes wrong in the brain to bring on dementia, they are investigating what goes right to stave off cognitive decline in select individuals. A small segment of the elderly population retains sharp memory even at age 80 and beyond, Changiz Geula of Northwestern University, Chicago, told this reporter at the SfN meeting. “What is special about these brains?” Hints emerged in a poster describing preliminary data from the university’s SuperAging study, headed by Geula. The findings thus far are based on postmortem analysis of five “super agers” 80 and above—three who performed like 50 year olds on standard neuropsychological tests, and two who showed stable cognitive stability for at least three years before death. Compared to control brain tissue from age-matched non-demented elderly, brains from the high-performing super agers had considerably lower numbers of tau tangles and pre-tangles in the entorhinal cortex, middle temporal gyrus, and cingulate cortex. On the other hand, super agers had greater numbers of amyloid plaques in these brain areas, relative to age-matched controls. Geula stressed that these data are very preliminary. He mentioned, for instance, that the tangle trend fades when data from the cognitively stable super agers are added to the analysis. Nevertheless, if reproduced with larger sample sizes, the new findings are intriguing because they suggest that super agers have particular characteristics that may help them compensate for the buildup of pathological amyloid. Identifying these factors is the long-term goal of the SuperAging project, Geula said. Education and other measures of cognitive reserve come to mind as possibilities (see ARF related news story), but Geula could not yet say whether these measures influenced the preliminary plaque and tangle findings. Forthcoming analyses should shed light on this issue, he said.—Esther Landhuis.

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DC: Therapeutic Stew—Various Morsels Savored at SfN

06 Dec 2008

One thing stood out at this year’s Society for Neuroscience annual meeting, held in Washington, DC, 15-19 November: researchers are not shy about putting new therapies through preclinical paces. While some of those potential therapies clearly aim to reduce what many consider the essence of AD pathology—Aβ and tau—other strategies, including those targeting inflammation, transcriptional control, and calcium toxicity, clearly added some spice to the mix.

One therapy that has been shown to lower brain Aβ in animals is polyphenol-rich grape seed extract (see ARF related news story). This palatable concoction may also help reduce tau toxicity, according to a presentation by Lap Ho from Giulio Pasinetti’s group at Mount Sinai Medical School, New York. Ho showed that a grape seed extract (GSE) helped rescue tau toxicity in various mouse models. In Tg2576 mice, phospho-tau241 immunoreactivity disappears and tau no longer forms fibrils when the animals take the extract with their food. In fly and mouse models of tauopathy, GSE effects were more distinct. Flies expressing the R406W mutant form of tau, which causes an inherited form of frontotemporal dementia, have a deformed eye phenotype; that is, their eyes are smaller and distorted. The extract partially rescued this defect, and the animals scored higher in visual screening tests. In the JNPL3 mouse, which expresses P301L mutant tau that causes frontotemporal dementia in other families, 150 mg/Kg/day of the extract (equivalent to about 800 mg/day in humans) rescued motor dysfunction and reduced fatality rates, Ho reported at SfN.

It appears that polyphenols in the extract may behave as general inhibitors of protein fibrillization. Ho showed that, in vitro, the grape seed extract reduced aggregation of a synthetic tau hexapeptide (VQIVYK), which serves to model tau aggregation. The GSE reduced thioflavin fluorescence resulting from aggregation of the hexapeptide, and results from photo-induced cross-linking of unmodified proteins (also known as PICUP) assays showed that the extract prevented peptide oligomerization. In a different presentation, Pasinetti lab collaborator David Teplow, University of California at Los Angeles, reported similar effects on Aβ. In thioflavin fluorescence and EM studies, Teplow found that the grape seed extract MegaNatural-AZ, a commercially available nutraceutical, prevented Aβ fibrillization, while PICUP assays also revealed reduced oligomerization. This work appeared in the 21 November Journal of Biological Chemistry (see Ono et al., 2008). Both Teplow and Ho used circular dichroism spectrometry to demonstrate that the extract prevented formation of β-sheet structures in Aβ and the tau hexapeptide, respectively, and that it eliminated paired helical formations of tau. The GSE even broke apart pre-formed tau aggregates, increasing their dissociation within 15 minutes and in a dose-dependent manner, reported Ho. Teplow showed that MegaNatural-AZ prevented cell death as judged by lactate dehydrogenase release and MTT (3-[4, 5-dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide) reduction, a measure of cell metabolic activity.

In addition to their anti-amyloidogenic potential, polyphenols also have anti-inflammatory activity, and this complicates the interpretation of their in-vivo effects (see ARF related news story). In fact, Yan-Jiang Wang, Flinders University, Adelaide, Australia, reported that GSE reduces not only Aβ but also inflammatory responses when given to three-month-old double transgenic mice (APPSwe/PS1dE9). At 12 months, Aβ deposits were down 45 percent in GSE-treated animals and microgliosis was down 70 percent, suggesting that at least some of the effects of polyphenols may be due to anti-inflammatory activity.

Anti-inflammatory agents have disappointed in AD clinical trials, though they continue to be screened in clinical and preclinical trials. One anti-inflammatory that has been considered is triflusal, which prevents activation of the inflammatory transcription factor NF-κB. Lidia Sereno from Teresa Gomez-Isla’s laboratory at the Universitat Autònoma de Barcelona, Spain, reported that when given to 10-month-old Tg2576 mice for three months, triflusal dampened both glial activation in the brain and expression of the proinflammatory markers IL-1β and TNFα. This happened alongside increased expression of brain-derived neurotrophic factor and c-fos, which is activated during learning. In behavioral tests, the animals performed better than untreated transgenic controls in a Morris water maze and contextualfear conditioning tests of learning and memory. Treated mice also had significantly reduced plaque load as determined by thioflavin S staining. The findings suggest that triflusal might have some potential as an AD therapeutic. Alas, a recent clinical trial run by Gomez-Isla and colleagues was halted prematurely due to recruitment problems (see ARF related news story and Gomez-Isla et al., 2008).

Non-steroidal anti-inflammatories have been given a fair shake in clinical trials, but one that has not drawn much attention is tolfenamic acid, currently approved in Europe for migraine. Lina Adwan from the University of Rhode Island, Kingston, reported that the drug might be worth studying as a potential AD therapeutic as well, though not because of its NSAID activity. Tolfenamic acid apparently reduces activity of the transcription factor Sp1 in pancreatic tumors. Because Sp1 has been implicated in regulation of APP expression, Adwan and colleagues examined the effect of the compound on APP levels. They found that in wild-type mice, tolfenamic acid reduced APP expression in the cerebral cortex and, slightly but statistically significantly, reduced Aβ40 levels in the brain as well. Treated guinea pigs showed a similar trend. It remains to be seen how this drug might work in AD mouse models and exactly how it affects Sp1 activity. “Indications are that it may activate a protease that degrades the transcription factor,” said Adwan.

Another signaling molecule that came in for scrutiny was macrophage colony stimulating factor (MCSF). Researchers led by Tony Wyss-Coray at Stanford University, Palo Alto, California, reported last year that MCSF was one of a panel of 18 blood markers that has diagnostic potential for AD (see ARF related news story). In Washington, Jian Luo, from Wyss-Coray’s lab, reported that in an independent analysis performed since then, MCSF plasma levels were significantly lower in AD patients (n = 85) than age-matched controls (n = 127). To see if increasing MCSF might help ameliorate AD pathology, Luo and colleagues administered this protein intraperitoneally to seven- to eight-month-old APP transgenic mice (Thy1-hAPP). Ten weeks later, the treated mice performed much better in the Morris water maze. Similarly, older mice (16-18 months) treated three times a week for four weeks performed better than controls. Curiously, these improvements occurred without any change in brain levels of soluble or insoluble Aβ, or in microglial activation.

To further probe the actions of MCSF, Luo and colleagues turned to the kainic acid excitotoxicity model. When administered 24 hours before kainite, MCSF almost completely blocked neuronal loss, measured five days later in the CA1 region of the hippocampus, and prevented loss of calbindin, used as a marker of neurodegeneration. Luo showed that kainate turned up activation of microglia, as judged by increased immunoreactivity to microglial markers CD68 and CD11b, but that this could be prevented by addition of MCSF. The numbers of microglia, on the other hand, were unchanged (determined by Iba1 immunoreactivity). Luo concluded by suggesting that MCSF might improve learning and memory performance in APP transgenic mice because it counteracts the excitotoxic effects of Aβ, making MCSF a potential future therapeutic for AD.

A further cytotoxin that has been linked to neurodegeneration in AD models is calcium (see related ARF live discussion). A poster by Dong Liu, from Mark Mattson’s lab at the NIA, described a novel way to reduce calcium toxicity by wedging open potassium ATP (K-ATP) channels. The researchers previously reported that diazoxide, a K-ATP channel opener, can protect against neuronal death caused by ischemia and apoptosis (see Liu et al., 2003). In Washington, Liu showed that diazoxide can improve learning and memory when chronically administered to 3xTg mice expressing human mutant APP, presenilin, and tau. The researchers began diazoxide treatment by adding the channel opener to the drinking water when the mice were four months old, and at 12 months tested their behavior. In both the Morris water maze test of learning and memory and in an open field test of spontaneous locomotor activity, the treated animals outperformed controls. Postmortem examination showed that treated animals had a dramatic reduction in Aβ levels in the hippocampus and, to a lesser degree, in the cortex. Levels of phosphorylated tau were also lower in treated animals.

Liu said that it is not clear how diazoxide works because it binds to both mitochondrial and cell membrane K-ATP channels. However, the fact that it hyperpolarizes cells and reduces calcium influx in hippocampal neurons suggests that dampening calcium cytotoxicity may be a crucial aspect. In the treated 3xTg mice, diazoxide also reduced oxygen consumption and increased cerebral blood flow, which might also be important factors in reducing pathology.

However tantalizing some of these approaches might seem, it should be kept in mind that promising preclinical strategies often fail miserably when applied to real-life disease. Trials of immunotherapies, vitamins, anti-inflammatories, statins, and most recently gingko biloba (see ARF related news story) are testament to the difficulties in translational research. As always, the real proof of the pudding will be in the eating.—Tom Fagan.

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References

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Paper Citations

Ono K, Condron MM, Ho L, Wang J, Zhao W, Pasinetti GM, Teplow DB. Effects of grape seed-derived polyphenols on amyloid beta-protein self-assembly and cytotoxicity. J Biol Chem. 2008 Nov 21;283(47):32176-87. PubMed.
Gómez-Isla T, Blesa R, Boada M, Clarimón J, del Ser T, Domenech G, Ferro JM, Gómez-Ansón B, Manubens JM, Martínez-Lage JM, Muñoz D, Peña-Casanova J, Torres F, . A randomized, double-blind, placebo controlled-trial of triflusal in mild cognitive impairment: the TRIMCI study. Alzheimer Dis Assoc Disord. 2008 Jan-Mar;22(1):21-9. PubMed.
Liu D, Slevin JR, Lu C, Chan SL, Hansson M, Elmér E, Mattson MP. Involvement of mitochondrial K+ release and cellular efflux in ischemic and apoptotic neuronal death. J Neurochem. 2003 Aug;86(4):966-79. PubMed.

DC: Magnifying Glass on Poisoned Mitochondria in ALS

10 Dec 2008

In the spinal cord, as in life, too much of a good thing can be detrimental. The copper chaperone CCS, which assists copper/zinc-superoxide dismutase (SOD1) to load its metallic co-factors, form an intra-subunit disulfide bond, and dimerize, is critical to stabilize the mature protein (for review, see Furukawa and O’Halloran, 2006). SOD1 mutations cause ALS in 20 percent of inherited cases of disease, so one might hypothesize that providing extra chaperone to help the mutant SOD1 fold properly would be beneficial. Yet Jeffrey Elliott, of the University of Texas Southwestern Medical Center in Dallas, and colleagues have shown that in mice carrying a mutant human SOD1, overexpression of CCS has a detrimental phenotype, sickening the mice within weeks as opposed to the months that it takes for symptoms to develop in a SOD1 single mutant. At the Society for Neuroscience annual meeting in Washington, DC, 15-19 November, first author Marjatta Son, who works with Elliott, presented a poster showing that different mutations in SOD1 react differently to CCS overexpression. Son found that the disulfide bond redox state of mSOD1 determines whether its toxicity is exacerbated by CCS.

The Elliott lab uses excess CCS as a sort of genetic magnifying glass to amplify the pathology of mSOD1 in mitochondria. Though SOD1 is mostly cytosolic, a small fraction is normally found in the mitochondrial intermembrane space, where it appears to aid mitochondrial manganese SOD2 in cleaning up reactive oxygen species (Sturtz et al., 2001). But some mSOD1 mice have mitochondrial pathology, including vacuolization of the mitochondria in many cell types (reviewed in Dupuis et al., 2004).

In yeast, CCS influences the cellular partitioning of SOD1 between the cytosol and mitochondria. When Son and Elliott overexpressed CCS in mSOD1 mice, “It had the most amazing effect on disease course,” Elliott said. CCS/SOD1-G93A mice develop tremors, ataxia, and spasticity at an average age of 11 days, compared to the six months it takes SOD1-G93A single mutants to exhibit symptoms (Son et al., 2007).

There are more than 100 distinct SOD1 mutations that have been linked to ALS, but only some, such as G93A and G37R, cause severe mitochondrial pathology. Other SOD1 mutations, such as G86R, have minimal vacuolization of mitochondria. Son used the CCS magnifying glass to probe the different mechanisms of four different SOD1 mutations: G93A, G37R, G86R, and L126Z. Both the G93A and G37R mutations responded strongly to additional CCS, making disease significantly worse in mice. Yet G86R and L126Z mice were not affected by overexpressed CCS.

Son looked to the redox state of mSOD1 as a potential explanation for the different phenotypes. In wild-type mice, approximately 15 percent of SOD1 has its disulfide bond in the reduced state. Elliott’s lab recently found that overexpressed CCS favors oxidation of the two cysteine amino acids that form the bond in wild-type SOD1, oxidizing 100 percent of the protein. However, extra CCS favors reduction of the same amino acids in SOD1-G93A (Proescher et al., 2008). Like SOD1-G93A, SOD1-G37R is also more likely to be reduced in mice overexpressing CCS (approximately 30 percent of SOD1 was reduced) than mutant SOD1 alone (10 percent reduced in young animals). In contrast, SOD1-G86R and SOD1-L126Z are fully reduced in single mutants and excess CCS does not affect their redox state, explaining why overexpressed CCS does not exacerbate disease in SOD1-G86R and SOD1-L126Z animals. Son and Elliott suggest that mSOD1’s redox state may be important in determining disease progression and pathology.

The explanation for the toxicity of disulfide-reduced mitochondrial SOD1 is still a mystery. Elliott hypothesizes that reduced SOD1 gums up the works by forming disulfide bonds with other essential mitochondrial proteins. Son and Elliott recently showed that CCS overexpression in SOD1-G93A mice results in lowered levels of cytochrome c oxidase (COX), which catalyzes the last electron transfer in the respiratory electron transport chain (Son et al., 2008). COX assembly is a delicate process requiring several proteins, Elliott said, so it’s possible that the reduced SOD1 interacts with and prevents those proteins from doing their job, cutting respiration and disabling the mitochondria.

Also at the SfN meeting, Elliott lab member Krishna Puttaparthi presented a poster outlining a screen to identify molecules that enhance COX levels. Puttaparthi dissected spinal cord slices from CCS/SOD1-G93A mice and cultured them with different treatments. Puttaparthi identified three compounds that increased COX levels. One such compound, resveratrol, increased survival by an average of 22 days when given to SOD1-G93A mice.

By amplifying CCS to exacerbate the mitochondrial phenotype of mSOD1 animals, Elliott and Culotta hope to zero in on the molecular mechanism of mitochondrial pathology in familial ALS caused by SOD1 mutations. For example, Culotta plans to use crystallography to collect “snapshots” of the incompletely developed SOD1 in cells overexpressing CCS.

The interactions between CCS and SOD1 are an example of how sensitive cells can be to protein levels. “You really need to have the right amount of CCS and the right amount of SOD1 to make the magic work,” Culotta said.—Amber Dance.

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References

Paper Citations

Furukawa Y, O'Halloran TV. Posttranslational modifications in Cu,Zn-superoxide dismutase and mutations associated with amyotrophic lateral sclerosis. Antioxid Redox Signal. 2006 May-Jun;8(5-6):847-67. PubMed.
Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC. A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J Biol Chem. 2001 Oct 12;276(41):38084-9. PubMed.
Dupuis L, Gonzalez de Aguilar JL, Oudart H, de Tapia M, Barbeito L, Loeffler JP. Mitochondria in amyotrophic lateral sclerosis: a trigger and a target. Neurodegener Dis. 2004;1(6):245-54. PubMed.
Son M, Puttaparthi K, Kawamata H, Rajendran B, Boyer PJ, Manfredi G, Elliott JL. Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology. Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):6072-7. PubMed.
Proescher JB, Son M, Elliott JL, Culotta VC. Biological effects of CCS in the absence of SOD1 enzyme activation: implications for disease in a mouse model for ALS. Hum Mol Genet. 2008 Jun 15;17(12):1728-37. PubMed.
Son M, Leary SC, Romain N, Pierrel F, Winge DR, Haller RG, Elliott JL. Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein. J Biol Chem. 2008 May 2;283(18):12267-75. PubMed.

Overworked HDACs Leave Transcriptional Posts to Push DNA Repair

10 Dec 2008

In discussions of what goes awry during neurodegeneration, the words genome stability, cell cycle, and DNA repair often come out in the same breath. Now, two Boston-based research teams have linked these activities to the same chromatin-modifying proteins—histone deacetylases (HDACs). Writing in tomorrow’s issue of Neuron, Li-Huei Tsai, Massachusetts Institute of Technology, and colleagues have identified HDAC1 deregulation as a common mechanism behind cell cycle re-entry and double-strand DNA breaks in an Alzheimer disease mouse model. Studying SIRT1, the mammalian homolog of the yeast HDAC Sir2, researchers led by David Sinclair at Harvard Medical School report in the 28 November issue of Cell that SIRT1 normally represses cell cycle-related and other mouse genes but redistributes to DNA breaks to promote repair. This, in turn, unleashes age-related transcriptional changes. The two studies highlight proper gene regulation as a key aspect of neuronal health. They underscore the potential for HDAC-targeted therapeutics, several of which showed preclinical promise at the Society for Neuroscience annual meeting held 15-19 November in Washington, DC (see ARF companion story).

When all is well in the brain, neurons are terminally differentiated cells whose replication machinery has remained dormant for decades. However, researchers have long observed that some neurons enter a mitotic frenzy that presumably leads to their early demise in AD (Vincent et al., 1996; Yang et al., 2003; and ARF related news story). Even so, considerable uncertainty has lingered as to whether the errant cell cycle activity causes or results from neuronal degeneration. The Neuron paper lays to rest this chicken-and-egg controversy, Tsai suggested in an interview with ARF. Cell cycle dysfunction “actually happens before you see any signs of neurodegeneration,” she said. “Our study really suggests that this is a very early event and likely contributes to neurodegeneration as opposed to just being a terminal event.”

To start off, first author Dohoon Kim and colleagues used microarrays to probe transcriptional changes that precede neurodegeneration in Tsai’s AD mice, which also develop tau pathology and learning defects upon induction of forebrain-specific p25 (Cruz et al., 2003). Among the 225 genes that were differentially expressed in induced transgenics versus uninduced controls, an unusually high number (65) encoded proteins involved in cell cycle (e.g. PCNA, Ki-67, cyclins A, B, and E) or DNA repair (e.g., Rad51, BRCA1, Chk1). The vast majority (63 of 65) were upregulated upon p25 induction. In subsequent RT-PCR and immunofluorescence studies of p25 mouse brain cells, the researchers validated the microarray findings by showing that p25 induction in fact activates abnormal cell cycle activity and produces DNA breaks.

More hints that these ominous features might share a molecular trigger came when the team found markers for DNA damage (phosphoserine 129 histone H2AX [γH2AX]) and cell cycle progression (Ki-67) co-expressed in more than 90 percent of examined neurons. HDAC1, already implicated in transcriptional repression of various cell cycle genes, came to the fore as a possible common culprit for these problems. Confirming their hunch, the researchers demonstrated a p25/HDAC1 interaction in induced p25 mouse forebrain tissue and in 293T cells transfected with either p25 or p35 (the physiological protein from which pathological p25 is cleaved). They found that HDAC1 prefers p25 to p35, interacting with the former to a 12-fold higher extent than the latter in 293T cells.

This curious observation, combined with mapping studies showing that p25 binding occurs in an N-terminal region of HDAC1’s catalytic domain, suggested that interaction with p25 could interfere with HDAC1’s normal activities. Indeed, both p25-transfected 293T cells and induced p25 hippocampal cells had lower HDAC1 activity than their untransfected or uninduced controls. The researchers also found that inhibiting HDAC1 increased double-strand DNA breaks and cell death, and that overexpression of HDAC1 relieved these problems in primary cortical neurons, compared with untreated cells. Trying to make a case for in vivo significance, they showed that striatal HDAC1 injections given to rats one day before an ischemic insult reduced DNA damage and numbers of dying cells by more than a third relative to animals treated with catalytically dead HDAC1.

Interestingly, in the immunofluorescence studies of p25 transgenic neurons, the DNA damage signal that showed up after two weeks of p25 induction disappeared with four subsequent weeks of p25 suppression. This suggests that “the early stage when we start to see DNA lesions is still a reversible window of time,” Tsai told ARF. “If you can do something during this window, then probably neurodegeneration can be prevented.”

SIRT1 Resembles Yeast Cousin Sir2 as Genome Stabilizer
For its part, Sinclair’s work paints a similar picture of HDACs as factors that relocalize to DNA lesions to initiate repair and, in doing so, stray from their typical gene regulatory functions. However, his team arrived at these conclusions by entirely different means. Their study’s point of departure was the longstanding observation that Sir2 proteins in yeast function primarily by stabilizing the genome. Interested in epigenetic effects during aging, first author Philipp Oberdoerffer wondered, for the mammalian Sir2 homolog SIRT1 (a class III HDAC), “Is this function conserved, and does it impact proper organismal function?”

Initial clues that aging-related stressors might disrupt the transcriptional silencing function of SIRT1 came from experiments in mouse embryonic stem (ES) cells, where SIRT1 associates with and represses highly repetitive DNA. When the researchers subjected the cells to oxidative stress (H2O2), the amount of SIRT1 bound to satellite repeats dropped, and transcription at these loci went up—an effect reproduced by the pan-sirtuin inhibitor nicotinamide and counteracted by expression of a SIRT1 transgene.

To extend these findings to protein-coding genes, Oberdoerffer and colleagues performed “ChIP on chip” (chromatin immunoprecipitation combined with a genome-wide promoter array) to identify SIRT1 target genes. In untreated ES cells, SIRT1 associated with promoters of a variety of genes, most prominently those involved in chromatin assembly, transcriptional repression, ubiquitin-regulated protein degradation, and cell cycle regulation. However, in H2O2-treated cells, SIRT1 associated with less than 10 percent of these genes, and its binding pattern no longer fit the observed functional groups, suggesting that oxidative stress shifted SIRT1 to random sites across the genome.

Taking after its yeast homolog, SIRT1 relocalizes from silent genes to DNA lesions in response to stressors, the researchers found. In fact, SIRT1 physically associates with double-strand break sites and helps bring the double-strand break repair crew where it needs to go, as recruitment of Rad51 (a key double-strand break repair protein) to DNA lesions was down in SIRT1-deficient ES cells. Knockdown of SIRT1 or pharmacological inhibition of its activity reduced both pathways of double-strand break repair—homologous recombination-mediated repair and, to a lesser extent, non-homologous end-joining. Consistent with its key role in DNA repair, SIRT1 seems to be important for maintaining overall genomic stability. When the researchers subjected wild-type and SIRT1-deficient ES cells to H2O2, and analyzed them for chromatid breaks, chromosomal fusions, translocations, and other chromosomal abnormalities, they found many more aberrations in the SIRT1-deficient cells.

To put these findings into an in-vivo context, the scientists turned to p53+/- mice. Missing one copy of this tumor suppressor gene, these animals frequently develop cancer when exposed to ionizing radiation because the irradiation-induced DNA damage disrupts their remaining p53 allele. Hence, survival and cancer rates provided a nice readout for testing SIRT1 manipulations. The researchers found that, as expected, boosting SIRT1 expression and/or activity increased survival rates and reduced cancer frequency in irradiated p53+/- animals. In normal mice, two-thirds of the SIRT1-bound genes whose repression lifted with aging (comparing five-month-old and 30-month-old mice) also were re-expressed in H2O2-treated ES cells. Furthermore, in animals engineered to overexpress SIRT1 in the brain in an inducible fashion, switching on SIRT1 delayed these age-related transcriptional changes.

Together, the in-vivo findings encouraged the scientists because they indicated that having more SIRT1 around doesn’t detract from either of its functions. “You maintain your genomic integrity and at the same time you have extra SIRT1 to take care of the DNA damage,” Oberdoerffer told ARF. “Based on our paper, you would say that in general, in a DNA damage-driven aging environment, it might be a good thing to have more SIRT1.” SIRT1’s benefits extend to neurodegenerative disease, too. Last year, Tsai, Sinclair, and colleagues published a study showing that SIRT1 wards off neurodegeneration in the inducible p25 transgenic mouse and in cell-based models for AD and amyotrophic lateral sclerosis (Kim et al., 2007).

Oberdoerffer is keen on seeing whether other chromatin modifiers such as methylases and other HDACs exhibit similar protective effects as SIRT1. This question could be hard to tackle, as isoform-specific differences likely abound. Whereas Oberdoerffer’s paper suggests that a specific sirtuin guards against aging-driven transcriptional changes, a recent report from Frank LaFerla’s group at the University of California, Irvine, showed that a pan-sirtuin inhibitor (nicotinamide) unexpectedly boosted cognition and reduced tau pathology in AD mice (see ARF related news story).

Still, hopes remain high that deacetylases and other agents of epigenetic change will prove their worth as therapeutic targets for aging and neurodegenerative disease. “If you silence a gene, you can at least in theory always reverse that,” Oberdoerffer told ARF. “If you have a good understanding of the epigenomic picture, then you might understand a little better the aging process, and where and how to interfere with it.” For a glimpse at several promising HDAC-targeted drug candidates, read Part 2 of this series.—Esther Landhuis

This is Part 1 of a two-part series. See also Part 2.

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References

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Paper Citations

Vincent I, Rosado M, Davies P. Mitotic mechanisms in Alzheimer's disease?. J Cell Biol. 1996 Feb;132(3):413-25. PubMed.
Yang Y, Mufson EJ, Herrup K. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. 2003 Apr 1;23(7):2557-63. PubMed.
Cruz JC, Tseng HC, Goldman JA, Shih H, Tsai LH. Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron. 2003 Oct 30;40(3):471-83. PubMed.
Kim D, Nguyen MD, Dobbin MM, Fischer A, Sananbenesi F, Rodgers JT, Delalle I, Baur JA, Sui G, Armour SM, Puigserver P, Sinclair DA, Tsai LH. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis. EMBO J. 2007 Jul 11;26(13):3169-79. PubMed.

DC: Developing But Debatable—Deacetylase Inhibitors for CNS Disease?

11 Dec 2008

While some neurodegenerative disease therapies focus on signature proteins—for example, amyloid and tau in the case of Alzheimer’s—a growing number are addressing more fundamentally what sets neurons up for self-destruction. Some of these approaches target histone deacetylases (HDACs), chromatin-compressing enzymes that promote transcriptional repression and have recently come into the spotlight as regulators of DNA repair, too. In the scenario emerging from two new studies, certain deacetylases keep neurons healthy by silencing a diverse set of genes, but will ignore these loci and move to DNA breaks to rally repair crews in times of stress (see ARF companion story). Extra deacetylase would thus help ensure sufficient resources for both essential roles. But one could also imagine the opposite situation, in which having more of a particular deacetylase might be a bad thing if, for instance, that HDAC repressed a gene whose upregulation helps treat or prevent a disease. In those cases, HDAC inhibitors could turn up winners. As reported in recent literature and at the Society for Neuroscience (SfN) annual meeting held 15-19 November in Washington, DC, several such compounds have passed muster in preclinical testing, and a few are moving toward human trials. At first glance, these findings seem to fly in the face of recent work suggesting protective roles for select deacetylases (see Part 1 of this two-part story). Considering the collective data, experts agree it is impossible to label HDACs as generally “good” or “bad” for aging and neurodegeneration, and highlight the importance of identifying their target genes and evaluating HDAC-targeting compounds for specific conditions.

In an SfN poster featuring data to be published in the journal Neuropsychopharmacology, researchers led by Alberto Pérez-Mediavilla and Ana Garcia-Osta of University of Navarra in Pamplona, Spain, showed that the HDAC inhibitor sodium 4-phenylbutyrate rescues cognitive deficits in Tg2576 mice, a widely used AD model that overexpresses mutant human amyloid precursor protein (APP) and develops memory impairment by nine to 10 months. Phenylbutyrate is an orally bioavailable short-chain fatty acid. By inhibiting deacetylases, it activates genes involved in regulating cell development and proliferation. It also appears to act as a molecular chaperone that reverses pathological protein aggregation in human diseases. A related compound, the HDAC inhibitor sodium butyrate, improved learning and long-term memory recovery in inducible p25 transgenic mice, which exhibit tau pathology, cognitive defects, and neurodegeneration (Fischer et al., 2007 and see ARF related news story).

Poster lead author Garcia-Osta and colleagues gave 16-week-old Tg2576 or non-transgenic mice intraperitoneal injections of 4-phenylbutyrate (200 mg/kg) or saline once a day for five weeks. The drug restored spatial memory in Tg2576 animals to wild-type levels in the Morris water maze with invisible platform and in 15- and 60-second probe trials. Treated Tg2576 mice showed no change in Aβ40 or Aβ42 levels or plaque load, but had reduced tau phosphorylation (with steady tau protein levels) and GSK3β activity, compared to wild-type and saline-injected controls. The effects on tau but not Aβ are intriguing, as a study published last month showed that an inhibitor of sirtuins (Class 3 HDACs) had similar tau-selective effects and improved cognition in a different AD mouse model (3xTg) (see ARF related news story). In the SfN study, phenylbutyrate treatment also enhanced expression of synaptic plasticity markers GluR1 and PSD95 in Tg2576 mice.

In experiments to tease out the compound’s specificity, the researchers found that cortical brain extracts from Tg2576 mice have reduced levels of acetylated histone 4 (H4) relative to non-transgenic animals, and that drug treatment partially restores this effect (but does nothing to levels of acetylated H3). In cultured primary neurons, drops in H3 and H4 acetylation in Tg2576 relative to wild-type cells were both reversed by four days of drug treatment. These data leave open the possibility that 4-phenylbutyrate may affect acetylase activity as well, Pérez-Mediavilla noted in an e-mail to ARF. He added that it is unclear at this point which HDACs are targeted by the drug.

The SfN meeting also featured preclinical data on an HDAC inhibitor that appears to enhance memory in normal mice. Developed by EnVivo Pharmaceuticals, a small biotech in Watertown, Massachusetts, the compound EVP-0334 can be taken orally and penetrates the brain well. As detailed on posters by Holger Patzke and Liza Leventhal, of EnVivo, and colleagues, EVP-0334 primarily targets Class 1, 2A, and 4 HDACs, inhibiting deacetylase activity in mouse cortical neurons and human astrocytes with IC50 values ranging from 0.3-1microM. In brain extracts from mice given the drug orally, histones 2A, 3, and 4 show increased acetylation with a minimal effective dose of 10 mg/kg. Wild-type mice receiving a similar dose had better short-term (90-minute) and long-term (24-hour) memory than did vehicle-treated controls in a novel object recognition test. “This is striking to us because the drug is completely gone from the mouse at 24 hours,” EnVivo scientist Michael Ahlijanian said at an SfN press briefing.

In a post-briefing conversation, he told reporters the compound has not been tested in any AD mouse models. He also could not say much about transcriptional profiles affected by the drug. “We can’t point to any single gene or group of genes that is responsible for these memory-enhancing effects,” Ahlijanian said. Nevertheless, the drug is moving toward the clinical pipeline. A Phase 1 trial focusing on pharmacokinetics and safety should start within a year, he told ARF.

HDAC inhibitors may also hold promise for treating Huntington disease, for which transcriptional dysregulation is emerging as a key pathology. Reporting in the 7 October issue of PNAS, Elizabeth Thomas and colleagues at the Scripps Research Institute in La Jolla, California, showed that oral administration of an HDAC3-prefering inhibitor improved motor performance, overall appearance, and body weight in an HD mouse model (R6/2300Q). The drug slowed brain atrophy and partially relieved gene expression changes caused by accumulation of mutant huntingtin protein in the striatum, cortex, and cerebellum. Repligen Corporation in Waltham, Massachusetts, is licensing this and another related compound for Friedreich’s ataxia, an inherited neurodegenerative condition that affects one in every 50,000 people in the U.S. The company “is also committed to pushing our compounds into trials for HD,” Thomas wrote in an e-mail to ARF. “The most optimistic time frame would be one year from now.”

Not to be left out, Parkinson disease may have some HDAC-based drug candidates as well. Last year, scientists reported that inhibitors of SIRT2 (a Class 3 HDAC) can prevent α-synuclein toxicity in fly and cell models of PD (Outeiro et al., 2007 and see ARF related news story). SIRT2 inhibition also appears to stave off neurodegeneration in a fly model of HD (Pallos et al., 2008).

HDAC-targeting compounds are certainly making a splash in CNS drug development. The global transcriptional effects of such drugs can be a two-edged sword, though—powerful if the right genes are changed in the right directions, devastating if not. “It’s very clear that different HDACs have very different functions,” said Li-Huei Tsai of Massachusetts Institute of Technology, whose study in today’s issue of Neuron proposes HDAC1 deregulation as a common culprit for cell cycle reactivation and DNA damage in p25 mice (see Part 1 of this two-part story).

Philipp Oberdoerffer, whose new work identified a deacetylase (SIRT1) as a joint mediator of genome stability and DNA repair (Oberdoerffer et al., 2008) agreed, but noted that specificity concerns need not overshadow a compound’s therapeutic promise. “These inhibitors have many, many effects, so if you happen to activate some genes that may be protective in a certain setting, that is already an achievement,” he wrote in an e-mail to ARF.

However, given his and Tsai’s recent studies, which suggest that certain HDACs themselves can be protective, Oberdoerffer cautions against using HDAC inhibitors to prevent neurodegeneration in healthy people. “In the long run, messing with transcriptional regulation and the role of HDACs in the DNA damage response may have adverse effects,” he noted.

To optimize the potential of HDAC-targeting compounds, efforts should be made to nail down their specificity, Tsai suggested. “It’s extremely important to know what you are dealing with. I would say that the way to go is to develop isoform-specific inhibitors. They are going to be more selective and therefore more potent and probably a lot safer,” she told ARF.—Esther Landhuis.

This is Part 2 of a two-part series. See also Part 1.

References

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Paper Citations

Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai LH. Recovery of learning and memory is associated with chromatin remodelling. Nature. 2007 May 10;447(7141):178-82. PubMed.
Outeiro TF, Kontopoulos E, Altmann SM, Kufareva I, Strathearn KE, Amore AM, Volk CB, Maxwell MM, Rochet JC, McLean PJ, Young AB, Abagyan R, Feany MB, Hyman BT, Kazantsev AG. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson's disease. Science. 2007 Jul 27;317(5837):516-9. PubMed.
Pallos J, Bodai L, Lukacsovich T, Purcell JM, Steffan JS, Thompson LM, Marsh JL. Inhibition of specific HDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease. Hum Mol Genet. 2008 Dec 1;17(23):3767-75. PubMed.
Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, Park SK, Hartlerode A, Stegmuller J, Hafner A, Loerch P, Wright SM, Mills KD, Bonni A, Yankner BA, Scully R, Prolla TA, Alt FW, Sinclair DA. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell. 2008 Nov 28;135(5):907-18. PubMed.

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DC: New γ Secretase Inhibitors Hit APP, Spare Notch

13 Dec 2008

Specificity is a big issue for drug makers as side effects due to unpredicted interactions can scuttle potential pharmaceuticals. In the case of γ-secretase inhibitors, the promiscuous nature of the enzyme, particularly its appetite for the Notch substrate, has produced its fair share of problems. In response, scientists have come up with a second generation of inhibitors. As reported at the annual meeting of the Society for Neuroscience, held 15-19 November in Washington, DC, and also at the International Conference on Alzheimer’s Disease last July in Chicago, drug companies have new compounds in the pipeline that appear to block γ-secretase cleavage of APP while sparing Notch processing. Two such compounds, from Wyeth and Bristol-Myers Squibb, have entered Phase 1 clinical trials.

Researchers at Wyeth are testing GSI-953, a thiophene sulfonamide derivative. At the SfN meeting, Steven Jacobsen at Wyeth Research in Princeton, New Jersey, reported that the compound preferentially inhibits processing of APP over that of Notch. Also called Begacestat, GSI-953 was described last month in the Journal of Medicinal Chemistry (see Mayer et al., 2008). In cell-free and cell-based assays, the inhibitor blocks production of Aβ42/Aβ40 with an EC50 of about 10-15 nM, which is more potent than the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester), reported Jacobsen. Begacestat is less potent for Aβ reduction than Eli Lilly’s LY411575 (see ARF related news story), but is more potent than LY450139, which is in Phase 3 clinical trials. However, GSI-953 excels in selectivity. Jacobsen showed that the Notch/APP EC50 ratio is about 16.8, which beats the 0.9 and 2.4 for LY411575 and LY450139, respectively. The data “suggests that GSI-953 selectively inhibits cleavage of APP while sparing Notch processing,” said Jacobsen.

Exactly how GSI-953 works is not entirely clear, but it competes with other compounds that bind near the γ-secretase active site. In Tg2576 transgenic mice it reduces Aβ by about 40-60 percent in the CSF and brain. It works rapidly; within 15 minutes reduction in brain Aβ is apparent, said Jacobsen. This, incidentally, demonstrates that Aβ has a very short half-life. The compound also reverses contextual-fear memory deficits. Wyeth has taken the compound into Phase 1 clinical trials (see ClinicalTrials.gov), and preliminary data suggest that it causes a dose-dependent decrease in plasma Aβ in humans, followed by a rebound. This is typical for γ-secretase inhibitors and likely means that the drug has engaged its target in the periphery. “This does not necessarily reflect Aβ levels in the brain, since Aβ levels were not increased in the brain of TgAPP mice,” said Jacobsen.

Speaking at ICAD, Charlie Albright, Bristol-Myers Squibb, Wallingford, Connecticut, reported the same rebound plasma Aβ dynamic in both animal models and in human volunteers with BMS-708163, his company’s new Notch-sparing γ-secretase inhibitors. This compound decreases CSF Aβ40 at a safe and tolerable dose in humans, Albright told the audience. It has an IC50 for APP cleavage of 0.3 nM compared to 58 nM for Notch. The compound dose-dependently lowers Aβ in the brain of rats and dogs and similarly reduces CSF Aβ40. Perhaps in keeping with the substrate specificity, the compound had no gastrointestinal, thymus, or spleen effects in animals treated with 10-fold the dose needed to see reduction in brain Aβ. Gastrointestinal and immune system problems have been documented in γ-secretase inhibitors in the past (see ARF related news story).

According to Albright, Bristol-Myers Squibb has also taken this compound into Phase 1 clinical trials, though ClinicalTrials.gov does not list an active trial at present. (It does list a completed drug interaction study.) At ICAD, Albright reported that up to 150 mg of the compound has been given to healthy volunteers for up to 28 days. Pharmacokinetics shows that the drug disappears from the plasma in a biphasic fashion, with a second phase half-life of about 40 hours, Albright said. The researchers used continuous lumbar catheterization in humans to measure changes in CSF Aβ. They saw a dose-dependent reduction in CSF Aβ after single and multiple doses of the compound. After the 28-day study, trough levels of CSF Aβ40 and CSF Aβ42 were reduced by about 30 percent at 100 mg and 60 percent at 150 mg doses.

When asked, Albright responded that there were no serious adverse events associated with this Phase 1 trial. Of course, further study will be needed to test the efficacy and safety of both these compounds. For an up-to-date review on γ-secretase inhibitors, see Imbimbo, 2008.—Tom Fagan.

References

News Citations

Paper Citations

Mayer SC, Kreft AF, Harrison B, Abou-Gharbia M, Antane M, Aschmies S, Atchison K, Chlenov M, Cole DC, Comery T, Diamantidis G, Ellingboe J, Fan K, Galante R, Gonzales C, Ho DM, Hoke ME, Hu Y, Huryn D, Jain U, Jin M, Kremer K, Kubrak D, Lin M, Lu P, Magolda R, Martone R, Moore W, Oganesian A, Pangalos MN, Porte A, Reinhart P, Resnick L, Riddell DR, Sonnenberg-Reines J, Stock JR, Sun SC, Wagner E, Wang T, Woller K, Xu Z, Zaleska MM, Zeldis J, Zhang M, Zhou H, Jacobsen JS. Discovery of begacestat, a Notch-1-sparing gamma-secretase inhibitor for the treatment of Alzheimer's disease. J Med Chem. 2008 Dec 11;51(23):7348-51. PubMed.

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