San Diego: Too Much APP Blocks Transport, Starves Down's Neurons

24 Oct 2004

Which genes are responsible for the known phenomenon that cholinergic neurons in Alzheimer disease and Down syndrome die because they can’t get their daily fix of nerve growth factor? APP is a natural suspect when it comes to shared features of Down and AD, and indeed, you need to look no further. Even so, you do need to think carefully about what fragment of APP may be the real culprit, as it might just be a lesser-known form called C99. That is the conclusion, and the main implication, respectively, from a presentation by Jean-Dominique Delcroix in Bill Mobley’s lab at Stanford University. Delcroix’ poster was one of 1,500 that kicked off the 34th annual meeting of the Society for Neuroscience, to be held in San Diego, California, for the next five days. A delight to the tireless, grueling to the compulsively comprehensive, the conference is expected to draw 30,000 participants this year. The Alzheimer Research Forum will post daily news stories for the next few weeks.

Delcroix’ poster is the latest chapter in a continuing story Mobley’s group has been developing over the past few years in an effort to explain the role of neurotrophic factors in neurodegeneration. The story begins with the realization that almost all Down patients eventually develop Alzheimer pathology, with the exception of a few who happen to miss those bits of their triplicated chromosome 21 that harbor the APP gene. People with Down's inherit three copies of chromosome 21, which carries the gene for APP, among many others. Indeed, this developmental disease has played a historic role in helping AD researchers nail APP as the first known AD gene, and it is considered a natural human model of AD. The physiological function of APP, however, remains enigmatic today.

Mobley’s group employed the Ts65DN mouse, a model for Down Syndrome that is trisomic for a segment of mouse chromosome 16 that houses 140 genes, including APP, and that is quite homologous to the corresponding stretch of human chromosome 21. Like aging people with Down's, and like AD patients, Ts65DN mice lose cholinergic neurons that project into the hippocampus as they age. Earlier, the researchers reported that these neurons wither because they are unable to transport the life-sustaining protein nerve growth factor back to the cell body after their nerve terminals have picked it up in the hippocampus (Cooper et al, 2001).

To find out which genes are behind this transport block, Delcroix worked with collaborators at Stanford and elsewhere to examine retrograde NGF transport in other mouse models. First, the researchers narrowed down the number of candidates with a second mouse strain that carries a smaller chromosome 16 triplication of 93 genes excluding APP. These mice have neither significant axonal transport defects, nor do their cholinergic neurons degenerate. The researchers confirmed with Q-PCR from laser-captured bits of hippocampal tissue that the first strain overexpresses APP but the second does not. The clincher came when the scientists bred the Ts65DN mice to heterozygous APP-deficient mice. Some of the offspring were triploid for all the genes of the chromosome 16 segment, but were diploid for APP. This restored axonal transport and prevented neurodegeneration.

Moreover, Delcroix and colleagues asked directly whether APP overexpression was sufficient to stall NGF transport. They assessed retrograde NGF transport in mice that express low levels of full-length human APP from a yeast artificial chromosome (Lamb et al., 1999), and found that the mice indeed had a transport reduction, though not enough to cause neurodegeneration.

So far the story is clear-cut, but it quickly gets murky, said Delcroix. Which form of APP impairs NGF transport, then? And does APP processing have something to do with it? It turns out that NGF transport in various AD mouse models varies in ways pointing to the β-secretase cleavage product C99. For one thing, Aβ levels did not influence NGF transport. For another, mice overexpressing the Swedish mutation of APP, as well as mice overexpressing APP and PS1, had decreased NGF transport, but mice overexpressing only PS1 instead saw their NGF transport increase. This could perhaps reflect the ready degradation of C99 by overly abundant PS1, removing it before it can make a mess of axonal transport, Delcroix speculated. This question needs to be sorted out.

Even so, the data so far suggest that APP somehow interacts with the retrograde axonal transport mechanism in the neurons that are among the earliest to degenerate in Alzheimer disease. Numerous other studies also point to derailed axonal transport as an early step in the pathogenesis of neurodegenerative diseases (see, for example, ARF related news story). APP is thought to function in axonal transport, though it is linked more strongly to anterograde transport (see ARF related news story). Finally, it’s worth noting that this research represents a case in point for a broader hypothesis about endosome signaling. It holds that transport on endosomes—in this case of NGF, its receptor TrkA, and related signaling proteins—subserves important signaling function, and that problems with this pathway are among the earliest signs of a neuron in distress (see Delcroix et al., 2004).—Gabrielle Strobel.

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Cooper JD, Salehi A, Delcroix JD, Howe CL, Belichenko PV, Chua-Couzens J, Kilbridge JF, Carlson EJ, Epstein CJ, Mobley WC. Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10439-44. PubMed.
Lamb BT, Sisodia SS, Lawler AM, Slunt HH, Kitt CA, Kearns WG, Pearson PL, Price DL, Gearhart JD. Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected]. Nat Genet. 1993 Sep;5(1):22-30. PubMed.
Delcroix JD, Valletta J, Wu C, Howe CL, Lai CF, Cooper JD, Belichenko PV, Salehi A, Mobley WC. Trafficking the NGF signal: implications for normal and degenerating neurons. Prog Brain Res. 2004;146:3-23. PubMed.

San Diego: HLA and γ-Secretase? A Curiosity Now, But Just You Wait…

24 Oct 2004

Venturing off the beaten path, Bryce Carey yesterday presented a poster at the 34th annual conference of the Society for Neuroscience about the interaction between an MCH class 1 protein and γ-secretase. The list of substrates for this key enzyme in amyloid production is growing continuously, but most of these substrates do not currently receive intense scrutiny for a possible role in the development of Alzheimer’s. This could change, however, if the notion becomes more firmly established that γ-secretase acts in AD pathogenesis not by a straightforward gain of function (i.e., Aβ production), but by a more subtle mix of partial gain and partial loss of function. Inklings of this trend pervade the field, and if it became more widely recognized, scientists will study the long list of γ-secretase targets with renewed interest to understand the broader role this proteolytic complex could have in AD and aging.

What’s with HLA, then? Carey, a technician in Dora Kovacs’s lab at Massachusetts General Hospital in Charlestown, began pursuing it when a sequence comparison showed that HLA-2A has an intramembrane domain shared by γ-secretase targets. But wait, you say—HLA is not a neuronal protein. True, it’s one of two classes of antigen-presenting membrane proteins, and its many varieties are traditionally thought to be expressed only on immune cells, where they present antigen to T cells to crank up an immune response. But a few years ago, Carla Shatz’s group at Harvard Medical School discovered to their surprise that developing, and indeed adult, mouse neurons express it, as well, and that lab is now studying class 1 MCH proteins for a possible function in the activity-dependent pruning that shapes the neonatal nervous system (see ARF related news story and ARF news).

In yesterday’s poster, Carey identifies the HLA-A2 as a substrate of α- and γ-secretase-mediated cleavage. He expressed HLA-A2 in CHO cells, and found that it undergoes ectodomain shedding when he also expressed ADAM-10, the leading candidate for the α-secretase role (see (see ARF related news story on Postina et al., 2004). This cleavage gives rise to a soluble piece and a membrane-anchored piece. The latter one then gets clipped further by γ-secretase, yielding a final soluble snippet that the cells quickly degrade, Carey’s poster suggests. Further experiments indicate that HLA-A2 forms a complex with β2-microglobulin, as indeed it normally does in lymphocytes. When Carey inhibited γ-secretase, less HLA was presented at the surface, hinting that γ-secretase cleavage might have to do with getting it there or be recycled properly.

If confirmed, one implication of this early work is that it could help explain why some γ-secretase inhibitor drugs interfere with T cell maturation (see ARF related news story). Furthermore, it might illuminate why some presenilin double knockout strains show changes in their thymocyte populations and mild autoimmune symptoms (see Tournoy et al., 2004). But most intriguing, perhaps, is speculation about what it might be doing in adult brain.—Gabrielle Strobel.

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San Diego: HIV and AD—Save the Body, Lose the Mind?

26 Oct 2004

Drug cocktails have transformed HIV infection from a deadly to a chronic disease, giving HIV carriers a new lease on life. But new long-term consequences of harboring the virus and of taking powerful drugs indefinitely keep cropping up, and the prospect of an Alzheimer’s-like dementia is the latest one. At the 34th meeting of the Society for Neuroscience here in San Diego, Cristian Achim of the University of Pittsburgh Medical School, and Lynn Pulliam of UCSF, yesterday and today presented early data suggesting that middle-aged people with HIV who are on highly active antiretroviral therapy (HAART) develop Aβ deposition, both inside neurons and as extracellular plaques. The two labs are working independently of each other.

It’s long been known that HIV infection will affect the brain at some point, but this is different, the scientists report. The brain can become a reservoir for the virus even while it is undetectable in blood and the HAART-treated patient shows no outward signs of disease. Accompanying HIV-associated risk factors for dementia include damage to the blood-brain barrier, infiltration of monocytes/macrophages (which most likely carry in the virus), and high levels of inflammatory cytokines. On top of that, a more specific risk for Alzheimer’s pathology may arise from two related processes, the scientists proposed. For one, these patients tend to develop high levels of insulin and insulin resistance, and this may have to do with interactions between the HIV drugs and the Aβ-degrading enzyme IDE, Achim’s work suggests. For another, an HIV protein called stat inhibits neprilysin, another enzyme that keeps Aβ levels down, reported Pulliam.

Achim described early data of an ongoing study in which he assessed amyloid deposition in postmortem tissue of 162 cases aged 25 to 70, with an average of 40, from the University of California Los Angeles and San Diego. These people had not had HIV encephalitis. Sixty percent of them had intraneuronal Aβ deposition confirmed by immunoelectron microscopy, and half of those also had extracellular deposits, mostly diffuse plaques, but no tangles. Achim hopes to follow a group of patients on long-term HAART forward with the PET amyloid imaging agent PIB to test how amyloid deposition correlates with cognitive performance over time.

Pulliam took a different tack by studying the effect of tat, an HIV protein secreted from infected macrophages and microglia, on neprilysin. Tat inhibited neprilysin in membrane preparations from human brain cultures and led to an increase in Aβ when added to cultures directly. Then she looked for Aβ in autopsy brain sections and, like Achim, found that middle-aged people with HIV had more plaques, large diffuse ones, than did controls. Plaque number correlated with how many years the person had been infected, but not with age. Whether this AD pathology leads to clinical Alzheimer disease is unclear, as is the question of how ApoE genotype influences the outcome. Even so, independent scientists at the conference agreed that long-term HAART-treated HIV survivors might constitute a new natural model of a form of Alzheimer’s.

This study will encourage researchers who propose that bacterial or viral pathogens cause some cases of sporadic Alzheimer’s see (Alzforum Discussion). This view is not widely held in the field, but it gained support from a presentation from Pat McGeer’s lab at University of British Columbia in Vancouver, Canada, reporting the cultivation of Borrelia burgdorferi spirochetes (the bacteria that cause Lyme disease) from Alzheimer brains.—Gabrielle Strobel.

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San Diego: Uncouplers Keep Mitochondria Burning Clean—Protect Neurons

30 Oct 2004

Next time you ponder the pros and cons of coal burning power plants, consider this: Mitochondria, our cellular power plants, do a great job keeping us supplied with ATP, but they, too, have emission problems. As electrons cascade down the mitochondrial respiratory chain, like water down a hydroelectric shaft, they can leak out of the inner membrane to partially reduce oxygen, forming such reactive oxygen species (ROS) as superoxide radicals and hydrogen peroxide. ROS, of course, damage proteins and nucleic acids, and have been implicated in aging and neurodegenerative disease, most notably Alzheimer (see ARF related news story) and Parkinson disease, which can be elicited by chemicals that damage the respiratory chain.

How can we prevent electrons from leaking? At hydroelectric dams there’s a simple solution to a similar problem. When water pressure builds, the flood gates are opened and the water harmlessly bypasses the turbine. Not surprisingly, nature has an equally simple solution for mitochondria—uncouplers.

Uncouplers were the focus of several presentations at the Society for Neuroscience meeting in San Diego this past week. Embedded in the mitochondrial membrane, they are so named because they uncouple the electron transport chain (the river upstream) from ATP synthase (the turbine), allowing electrons to flow rapidly to their final destination, cytochrome c oxidase. Uncouplers minimize leakage, and hence ROS production, allowing the vast majority of electrons to end up in one of the most harmless molecules of all, water. Zane Andrews and colleagues working at Tamas Horvath’s lab at Yale University reported that expression of uncoupler protein 2 (UCP2) regulates the amount of mitochondrial ROS formed, and can protect dopaminergic neurons of the substantia nigra, which are the very cells that are damaged in Parkinson disease.

Andrews made transgenic mice that either overexpressed UCP2 or had it knocked out completely (see Meeting Abstract No. 903.13). To measure ROS produced by mitochondria in vivo, he perfused the animals with a single tail injection of dihydroethidium, which is converted to the fluorescent ethidium in the presence of superoxide. When Andrews used fluorescent microscopy to examine mitochondria isolated from dopaminergic neurons, he found that the amount of ethidium, and hence superoxide, was substantially reduced in the mice overexpressing the uncoupler, while in the UPC2 knockout animals, it was substantially higher than wild-type. In addition, overexpression of UCP2 increased respiratory chain uncoupling as judged by the amount of oxygen consumed by the cells.

But could uncouplers be exploited to reduce production of ROS and protect dopaminergic cells of the substantia nigra? Apparently so. When Andrews challenged the animals with MPTP, a mitochondrial toxin that achieved notoriety in the 80’s when it was found to be responsible for inducing Parkinson disease in heroin addicts, he found twice as many neurons in the substantia nigra of animals overexpressing the uncoupler than were found in wild-type animals also treated with the toxin. In addition, MPTP-treated UPC2 knockouts had 40 percent fewer neurons than did wild-type animals. The findings indicate that uncoupling mitochondria can provide some protection in a chemical simulation of Parkinson disease.

This work was supported by research from Bruno Conti at Tamas Bartfai’s lab at the Scripps Research Institute (see Meeting Abstract No. 94.9). Conti also described overexpression of UCP2, but this time under the control of the tyrosine hydroxylase promoter, which restricted expression of the transgenic protein to dopaminergic cells. Conti first showed that the additional UCP2, twice as much as found in wild-type animals, appeared only in the substantia nigra and locus coerulus, the latter also being affected in PD. Conti found that oxygen consumption was increased in animals overexpressing the uncoupler, and this was accompanied by a decrease in ROS. Lipid peroxidation and protein carbonylation, two measures of oxidative damage, were also reduced, and the transgenic animals were protected from MPTP—about 60 percent more neurons survived in transgenic animals than in controls.

Any attempt to use uncouplers as a therapeutic will have to be carefully controlled because uncoupling the respiratory chain completely would be lethal. But there may be subtle ways to modulate the electron transport chain and keep mitochondria running “cleaner.” Andrews reported, for example, that fatty acids (in this case palmitate) led to about a twofold, significant increase in electron transport chain uncoupling, suggesting that dietary modulation may be beneficial to our mitochondrial health, and ultimately, to us. Alzforum readers are no strangers to debate about caloric restriction and mitochondrial function, both of which are implicated in aging and the pathology of neurodegenerative diseases.—Tom Fagan.

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San Diego: Treating Forgetfulness—Triple Transgenics Provoke

03 Nov 2004

A mouse model that mimics both signature pathologies of Alzheimer disease develops a day-to-day forgetfulness at four months, a young adult age when the animals' brains accumulate the Aβ peptide inside their neurons but don’t yet harbor plaques or tangles. What’s more, a treatment proffered for years by an Israeli scientist appears to reverse this forgetfulness and both pathologies. If these findings hold up, they will stand out from among the more than 900 AD-related presentations at the 34th annual conference of the Society for Neuroscience, held last week in San Diego, California.

The mice in question are Frank LaFerla’s triple transgenics (3xTg-AD), which carry clinical mutations in the human APP, presenilin 1, and tau genes (see Oddo et al., 2003). These mice have generated widespread attention; indeed, LaFerla’s laboratory at University of California, Irvine, has responded to continuing requests from investigators by breeding and shipping the mice to investigators from nearly 20 countries and all across the United States. At the same time, other researchers caution that the model is artificial in its own way, as no humans are known to carry mutations in all three genes. Humans also do not have such high levels of overexpression as do most other AD mouse models.

In San Diego, the LaFerla lab reported their analysis of these mice in regard to lipids, oxidative stress, calcium channels, microglial activation, inflammation, and other aspects in 12 separate presentations. This news summary will focus on three of those dealing with cognition and treatment.

Lauren Billings described her search for a molecular change that marks the earliest cognitive deficit in the mice. She used the Morris water maze, a hippocampal task, and a fear-conditioning task testing function of the amygdala. At two months, the mice learned and remembered normally, indicating that the mice were not born with cognitive impairments, and whatever deficit they developed later was progressive and related to the transgenes. An ongoing longitudinal study has reached the 12-month point to date.

At six months, the 3xTg-AD homozygous mice needed more time to learn the task at hand, but they did learn it. An intriguing clue emerged when Billings broke down the data from this group effect to a trial-by-trial basis. The mice had four training trials per day, spaced apart by 30 seconds. The wild-type controls remember what they’ve learned the day before and therefore start off at a higher performance level every subsequent day of training. The 3xTg-AD also learn from test to test on a given day but by the next day have forgotten and repeat their learning curve from the day before. Compared to previously described learning deficits in AD-related mouse strains, this early result is a seductive one because it echoes a memory problem well known to AD clinicians, where a patient will remember a standard word list for a few minutes, but an hour later has no recollection of it at all.

But is this truly the earliest deficit in this mouse strain? Probably not, the scientists reasoned, because six-month-old homozygous animals showed a deficit during the 1.5-hour time course plus one from day to day, whereas hemizygous animals at this age were defective only in the latter measure. This meant that perhaps younger homozygous animals have only this 24-hour deficit, and indeed the four-month-olds did.

At this age, the researchers can detect only intraneuronal Aβ, not yet plaques or tangles. “This is correlation. We want to be able to say cause,” LaFerla said. The scientists then injected an anti-Aβ antibody (the monoclonal 6E10, which recognizes amino acids 1-17 of human Aβ) into the mice’s third ventricle, and found that it not only clears away intraneuronal pathology from the hippocampus, but also rescues the memory retention deficit. This worked only for the water maze task, however, not for the fear-conditioning task, as the pathology was not cleared in this brain region. This finding may reflect the amygdala’s distance from the injection site, the researchers assume.

“The bottom line is that early cognitive deficits follow closely with intraneuronal Aβ,” LaFerla said. “My personal belief is that the first cognitive deficits in AD are a functional change, not structural. At the point where memory retention first declines, there is not yet loss of synapses, no plaques, no tangles, no dystrophic neurites, no inflammation. Those all come later.”

When asked about the relevance of their model, LaFerla and Salvatore Oddo noted that it did predict accurately some observations of the AN-1792 trial, namely that it failed to remove mature tangles but appeared to reduce soluble tau (unpublished, but see Ferrer et al., 2004). They say this jibes with their recently published data (see ARF related news story), which supports the hypothesis that Aβ accumulation leads to tau pathology in vivo.

Oddo’s talk in San Diego builds on his prior finding that injection of an anti-amyloid antibody clears extra- and intraneuronal Aβ. This again highlights the open question of whether intraneuronal Aβ accumulation contributes to the extracellular plaques (see ARF Live Discussion and ARF Philadelphia news story). To address it, Oddo injected antibody into the mice’s brains once and analyzed the brains not days and weeks later, as in their August paper, but 6, 12, and 18 hours after the shot. Again, he saw that extracellular Aβ disappears first, followed by intraneuronal Aβ. Hours later, the intraneuronal Aβ aggregates first return, then the extracellular ones. This implies that the two pools are connected by a dynamic equilibrium. The finding that intracellular Aβ reappears before extracellular Aβ suggests that intraneuronal accumulation may be a precursor to the plaques, said LaFerla. Other scientists were impressed by this study, but noted that they would like to see follow-up work formally rule out the possibility that the intraneuronal Aβ represents the Aβ sequence within APP.

Last but not least, these data establish a basis on which to test the prowess of potential treatments, and this was the topic of the Antonella Caccamo’s poster. Caccamo injected into the 3xTg-AD mice’s intraperitoneal cavity—every 24 hours over two months, a tiring total of 2,800 times—the compound AF267B. Aficionados of the field may recognize it by its name as one of Abraham Fisher’s. Fisher and his colleagues, at the Israel Institute for Biological Research in Ness Ziona, have synthesized and studied series of small molecules in an effort to prove Fisher’s hypothesis that agonists that are highly selective for M1 muscarinic acetylcholine receptors could treat the symptoms of AD, as well as change its course, (see, for example Fisher et al., 2003 and Fisher et al., 1998). Some muscarinic agonists have been tested for years in vitro and in humans, but none have made it all the way to a useful AD drug. Some early muscarinic agonists have failed in clinical trials. That has made Fisher’s quest seem quixotic to some, despite his insistence that the failure was due to the compound's inadequate M1 selectivity and poor pharmacokinetics. Caccamo put Fisher’s hypothesis to the test, and her data appear to vindicate him. “Everything Abe Fisher has written has come true in our study,” LaFerla said.

Caccamo presented immunocytochemistry data suggesting that AF267B diminished the mice’s plaque pathology, intraneuronal pathology, and tau pathology in cortex and hippocampus. ELISA and Western blots also indicate decreases in soluble and insoluble Aβ formation. The Western blot shows a decrease in C99 (the product of β-secretase cleavage) and an increase in C83 (the APP fragment released by α-secretase cleavage.) Measuring steady-state levels, Caccamo and colleagues found a decrease in BACE, an increase in ADAM-17, and no change in ADAM-10 (see ARF related news story). AF267B also diminished phospho-reactive tau as stained with the antibody AT8. Finally, the compound reversed the memory retention deficit in the water maze.

“This is the first in-vivo evidence for Abe’s prediction of how this compound would shift APP processing and affect tau,” said LaFerla. At the Neuroscience meeting, and also in Neurobiology of Disease this month (see Farias et al., 2004), Fisher and colleagues at the Catholic University of Chile in Santiago laid out a mechanism for how this compound might counteract Aβ toxicity. In short, they propose that AF267B, via activation of the M1 receptor, inhibits the tau kinase GSK3-β and restores a downregulation of the wnt signaling pathway caused by Aβ.

Many questions remain. One fly in the ointment is that AF267B reversed neither the fear-conditioning deficit nor AD pathology in the amygdala. LaFerla hopes that an ongoing collaboration with memory researcher James McGaugh, also at UC Irvine, will shed light on this issue. Moreover, it's not yet clear that AF267B will meet the brain penetration and pharmacological requirements to become an AD drug.

Even so, this early data proposes to show for the first time a small molecule that can cross the blood-brain barrier, is bioavailable, and reverses Aβ and tau pathology as well as some behavioral deficits at doses that cause no adverse effects in mice. A biotechnology company in California has licensed the compound.—Gabrielle Strobel.

References

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Paper Citations

Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003 Jul 31;39(3):409-21. PubMed.
Ferrer I, Boada Rovira M, Sánchez Guerra ML, Rey MJ, Costa-Jussá F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol. 2004 Jan;14(1):11-20. PubMed.
Fisher A, Pittel Z, Haring R, Bar-Ner N, Kliger-Spatz M, Natan N, Egozi I, Sonego H, Marcovitch I, Brandeis R. M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer's disease: implications in future therapy. J Mol Neurosci. 2003;20(3):349-56. PubMed.
Fisher A, Brandeis R, Chapman S, Pittel Z, Michaelson DM. M1 muscarinic agonist treatment reverses cognitive and cholinergic impairments of apolipoprotein E-deficient mice. J Neurochem. 1998 May;70(5):1991-7. PubMed.
Farías GG, Godoy JA, Hernández F, Avila J, Fisher A, Inestrosa NC. M1 muscarinic receptor activation protects neurons from beta-amyloid toxicity. A role for Wnt signaling pathway. Neurobiol Dis. 2004 Nov;17(2):337-48. PubMed.

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San Diego: γ-Secretase Takes Scientists on a Wild Ride

08 Nov 2004

The more scientists learn about γ-secretase, the crazier it looks to them, according to Todd Golde of the Mayo Clinic in Jacksonville, Florida. Golde is but one of many avid students of this eccentric enzyme, which slices the APP protein twice inside the membrane to release the much-studied Aβ peptide and the little-studied C-terminal AICD fragment. Setbacks and complications notwithstanding, many academic and company researchers still scrounge the APP cleavage process for chances to interfere therapeutically, and consequently, γ-secretase was the topic of several dozen presentations at the 34th annual meeting of the Society for Neuroscience, held last week in San Diego. Here we present selected highlights.

Christian Haass, from Munich’s Ludwig-Maximilians-University, Germany, reported on his group’s efforts to understand the steps by which the γ-secretase complex comes together and matures. Remember, in addition to presenilin, the complex comprises at least Pen-2, nicastrin, and Aph-1 (see report from the recent Swiss Society of Neuropathology meeting).

Haass and other researchers have reported that maturation of the complex takes place in the endoplasmic reticulum (see, for example, ARF related news story). Might specific ER retention signals hold the proteins there until their partners have arrived? Haass reported that presenilin 1 (PS1) indeed has an ER retention signal in its C-terminus (SfN abstract 264.4). A CD4 chimeric protein having the PS1 C-terminal end was retained in the ER, for example, while a chimera with the PS1 N-terminal end migrated rapidly to the cell membrane. Postdoctoral fellow Christoph Kaether mutated the C-terminal end of PS1 to locate this retention signal. It turned out to be quite long, but it overlaps with the Pro-Ala-Leu-Pro stretch of amino acids that is found in all other presenilins. When the Kaether and Haass mutated this motif, PS1 still formed a complex with its three partners, but it moved rapidly out of the ER and it lacked γ-secretase activity. In contrast, when they tacked the wild-type retention signal on nicastrin, the complex assembled and was active. The results suggest that the retention signal keeps the partners of the complex in the ER long enough for maturation to occur. Haass presented evidence that maturation is accompanied by a masking of the retention signal, triggering migration of the secretase to the cell membrane (part of this work is in press in the EMBO Journal, Kaether et al.).

Despite this complex process of maturation, several labs have succeeded in reconstituting γ-secretase in other systems, for example, yeast (Edbauer et al., 2003). In San Diego, Takeshi Iwatsubo’s group at the University of Tokyo in Japan offered another system for study by describing a new method to express human γ-secretase components in budded baculovirus particles (Hahashi et al., 2004 and SfN abstract 90.10).

By contrast, Patrick Fraering, working with Michael Wolfe and Dennis Selkoe at Harvard Medical School in Boston, took a conventional biochemistry approach to studying this enzyme complex. He described how developing a six-step protocol for purifying human γ-secretase (Fraering et al., 2004) has enabled him to analyze directly the role of γ-secretase interactors and modifiers.

Using APP’s C99 fragment as a substrate (i.e., a recombinant protein consisting of the &β-CTF portion of APP), Fraering first used his in-vitro assay to test how lipids and membrane-perturbing detergents influence γ-secretase cleavage. Sphingomyelin and phosphatidylcholine increased the enzyme’s activity but not the ratio of Aβ42/40 products, whereas the detergents CHAPSO and SDS did change that ratio. The results suggest that the precise cleavage specificity of the enzyme is sensitive to lipid conditions. Several other groups are studying how membrane lipids alter APP processing and, in turn, how various forms of Aβ itself might compromise the integrity of membranes (see ARF related news story).

Next, Fraering used this system to reproduce aspects of a study from Paul Greengard’s laboratory, which had suggested that the approved cancer drug Gleevec selectively inhibits γ-secretase cleavage of APP while leaving Notch alone (Netzer et al., 2003). This paper seemed to revive γ-secretase as a drug target, an idea that had taken a beating as the list of γ-secretase substrates grew and early inhibitors proved toxic (see ARF related news story). Tantalizing as the Greengard paper was, it also seemed puzzling. It purported that Gleevec, a card-carrying tyrosine kinase inhibitor, did not affect γ-secretase through this primary function, but instead worked by a different, unknown mechanism, possibly one having to do with the ATP requirement for Aβ production. How could such a mechanism enable Gleevec to shift specifically the cutting of APP but not Notch? The final answer is still not in, but Fraering used the purified human enzyme complex to confirm the Netzer et al. data. His data indicate that Gleevec acts directly on the γ-secretase. He showed that ATP itself can modulate the cleavage activity of the purified γ-secretase, and noted that a screen of 50 tyrosine kinase inhibitors brought up a few others that also block Aβ but not Notch cleavage, perhaps via an ATP binding site. (SfN abstract 264.14).

“This is the most convincing demonstration yet that you can distinguish between APP and Notch cleavage,” said Golde. This does not mean that a drug is at hand. The doses needed would be too toxic for chronic use in humans; besides, Gleevec does not cross the blood-brain barrier well. Moreover, APP cleavage modulated by Gleevec still increases APP C-terminal fragments, which themselves might be toxic, Golde added.

One possible reason why small molecules may influence APP cleavage but not that of Notch may be that they don’t bind to the active site of γ-secretase, but instead influence the enzyme-substrate interaction in other ways. In San Diego, Wolfe suggested APP processing may require two independent steps of docking followed by cleavage (see SfN abstract 264.1)

His evidence centers on the use of a classic tool in enzymology, i.e., transition-state analogues that bind to the active site of γ-secretase. His group also designed small, 10- to 13-residue peptides to mimic the 3D structure of the section of APP that protease cleaves. To keep these peptides in their correct α-helical shape, Wolfe and colleagues substitute α-methyl alanine (Aib), which favors the helical conformation, for selected APP amino acids (see Bihel et al., 2004). Using photoaffinity labeling, Wolfe showed that these Aib APP peptides bind to both the N- and C-terminal fragments of presenilin 1, though preferentially to the N-terminus. They also inhibit γ-secretase potently in cell-free systems.

But it was when Wolfe and his colleagues tried to use transition-state analogs to prevent these peptides from binding to presenilin that they could show the two types of compounds bound to distinct locations. Unexpectedly, one helical peptide, D10, bound to presenilin even in the presence of transition-state analogs (TSA), while another one that’s three amino acids longer did compete with the TSAs. The Wolfe lab suggested the following explanation: The shorter peptides bind only to the APP docking site on presenilin, which may be between two of the transmembrane helices that make up the barrel-shaped protease. The longer peptide not only binds to the docking site, but also protrudes sufficiently through the presenilin helices to access the active site that is on the inside of the barrel. In support of this theory, Wolfe noted that FAD mutations in presenilin substantially alter TSA-binding, but have little or no effect on D10 binding.

This research also ties in with Golde’s and Eddie Koo’s own collaborative work. They were the first to discover that certain NSAIDs tweak γ-secretase activity in a way that shifts APP cleavage away from Aβ42 production while not touching other substrates. In San Diego, Thomas Kukar from Golde’s lab told the audience that he has performed a broad search for other compounds that do this more potently than the initially discovered agents, ibuprofen and indomethacin (SfN abstract 264.13). This search turned up a surprising variety of compounds that raise Aβ42 production in cell-based assays, including some COX-2-inhibiting NSAIDs such as Celecoxib. Ironically, Vioxx, which was retracted this fall (see ARF related news story), does not raise Aβ42.

But no COX-2 inhibitor lowered Aβ42. In fact, when the scientists made chemical derivatives of a given Aβ42-lowering NSAID to turn it into a COX-2 selective compound, they found that the derivative now raised Aβ42 production. This prompted Kukar to claim that COX-2 inhibition and Aβ42-lowering are mutually exclusive functions on a given molecule. Moreover, in collaboration with Koo, Golde, and Christian Haass, Sarah Sagi from University of California, San Diego, reported that NSAIDs that can alter the cleavage site in APP, including sulindac sulfide and flurbiprofen, have no effect on the cleavage site for Notch or CD44, indicating that the NSAID effect is substrate-specific (see SfN abstract 264.10).

One of the most potent Aβ42-raising agents Kukar found is the PPARγ agonist and lipid regulator fenofibrate. Others are isoprenoid compounds that are dietary metabolites. In principle, this raises the question of whether some dietary isoprenoids, or compounds that somehow alter isoprenoid metabolism, could pose an environmental risk for AD, the scientists said. It is clearly too early to say, but this pathway should be studied, they add. Finally, Kukar addressed the question of how the compounds act. To do so, they tried to confirm a recent paper (Zhou et al., 2003) that suggested that the effect is indirect and mediated through the Rho-Rock pathway of small GTPases. Kukar and colleagues were unable to reproduce these data. He instead suggested that these modulators act on γ-secretase directly, not via a second messenger pathway, as did Fraering with regard to Gleevec.

Another development γ-secretase aficionados follow closely is that of the mysterious epsilon cleavage. Two years ago, German researchers reported that they found yet another cleavage at play in the sequential digestion of APP, and termed it epsilon (Weidemann et al., 2002). It occurs closer to the cytoplasmic side of the membrane than the γ cleavage and gives rise to AICD. Since then additional groups have tried to understand the relationship of this new cleavage with the ones already known, particularly γ. In San Diego, two groups presented new data indicating that the epsilon cleavage can precede γ cleavage of APP (see SfN abstracts 90.14 and 146.4.) This is intriguing in part because it would open up an even more upstream opportunity of interfering with Aβ generation than is being targeted currently.

Clearly, this bewildering enzyme complex is only beginning to yield its secrets. Even so, many scientists at the Neuroscience meeting shared a sense that there may yet be a workable drug strategy in it, eventually.—Gabrielle Strobel and Tom Fagan.

References

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Paper Citations

Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Reconstitution of gamma-secretase activity. Nat Cell Biol. 2003 May;5(5):486-8. PubMed.
Hayashi I, Urano Y, Fukuda R, Isoo N, Kodama T, Hamakubo T, Tomita T, Iwatsubo T. Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles. J Biol Chem. 2004 Sep 3;279(36):38040-6. PubMed.
Fraering PC, Ye W, Strub JM, Dolios G, LaVoie MJ, Ostaszewski BL, Van Dorsselaer A, Wang R, Selkoe DJ, Wolfe MS. Purification and characterization of the human gamma-secretase complex. Biochemistry. 2004 Aug 3;43(30):9774-89. PubMed.
Netzer WJ, Dou F, Cai D, Veach D, Jean S, Li Y, Bornmann WG, Clarkson B, Xu H, Greengard P. Gleevec inhibits beta-amyloid production but not Notch cleavage. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12444-9. PubMed.
Bihel F, Das C, Bowman MJ, Wolfe MS. Discovery of a Subnanomolar helical D-tridecapeptide inhibitor of gamma-secretase. J Med Chem. 2004 Jul 29;47(16):3931-3. PubMed.
Zhou Y, Su Y, Li B, Liu F, Ryder JW, Wu X, Gonzalez-DeWhitt PA, Gelfanova V, Hale JE, May PC, Paul SM, Ni B. Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science. 2003 Nov 14;302(5648):1215-7. PubMed.

San Diego: ApoE Effects Explored

10 Nov 2004

ApoE, the major lipoprotein of the brain, is one of the strongest risk factors for late-onset Alzheimer disease (AD), a fact that no doubt spurred the ApoE minisymposium at the 34th annual meeting of the Society for Neuroscience in San Diego last month.

Studies on ApoE have exploded since it was discovered that those carrying one copy of ApoE4 allele are more likely to get the disease than those with only ApoE2 or ApoE3—having two copies of ApoE4 puts one at even greater risk. But exactly how the lipoprotein increases the likelihood of getting AD has not been nailed down. There are plenty of theories (see ARF related news story), one being that it interferes with the processing of amyloid precursor protein (AβPP) and the production of amyloid peptides (Aβ). In fact, Mike Irizarry, Massachusetts General Hospital, Boston, has recently published data showing that all variants of the cholesterol transporter inhibit γ-secretase cleavage of APP, leading to reduced production of Aβ and accumulation of APP C-terminal fragments. Irizarry and colleagues had also found that ApoE reduces transcriptional activation mediated by the partnership of APP intracellular domain (AICD) and the transcriptional coactivator Fe65 (see Irizarry et al., 2004). But curiously, when they had examined the effect of ApoE on processing of Notch, another substrate of γ-secretase, they found little effect. So by what mechanism might the lipoprotein affect γ-secretase cleavage of APP?

This was the question Irizarry addressed. One possibility is that binding of ApoE to its receptor—LDL receptor related protein (LRP)—triggers some event that specifically modulates cleavage of APP by γ-secretase. To test this, Irizarry incubated cells with ApoE in the presence or absence of the LRP blocker RAP (he used a variety of cell types including primary cultured neurons). He found that Aβ production was attenuated under both conditions. In fact, even in cells devoid of LRP, ApoE affected APP processing. Next, Irizarry tested if ApoE might be involved in redistribution of cholesterol in the cell, a process that has been implicated in modulation of γ-secretase (see ARF related news story). When he measured efflux of cholesterol from cells incubated with ApoE, he found only a very small effect; ApoE2, for example, caused about a twofold increase in cholesterol efflux. However, similar amounts of high-density lipoprotein caused a 15-fold increase in cholesterol efflux without affecting APP processing, so the slight cholesterol efflux evoked by ApoE2 is unlikely to explain the effect on APP.

Sticking with the cellular distribution theme, Irizarry then looked to see if ApoE might affect the localization of APP. To test this, he used an antibody to the C-terminal of APP to locate the protein in the cell, finding that addition of ApoE caused the precursor to shift from the cell surface to intracellular granules. The location of presenilin 1, one of the major proteins of the γ-secretase complex, was unaffected. The results suggest that ApoE does not have a direct effect on γ-secretase, but instead keeps the precursor protein and the protease separated.

Yadong Huang, University of California at San Francisco, has slightly different ideas on the link between ApoE and AD. Huang has hypothesized that ApoE cleaving enzyme or, AECE, may play a major role in neurotoxicity because it cleaves ApoE near the C-terminus, leaving a truncated protein that is neurotoxic (see ARF related news story from the Institute for the Study of Aging symposium on ApoE in July 2003).

AECE, it turns out, is a chymotrypsin-like serine protease which can cleave ApoE in various locations including at leucine 268 and methionine 272. To determine what impact these different cleavages may have, Huang’s lab have made several truncated ApoE constructs and expressed them in mice. Animals expressing the protein truncated at amino acid 271 show age-related neurodegeneration and loss of CA3 neurons in the hippocampus. The protein co-localizes with synaptophysin, suggesting that it is present in the presynapses, but it is not found near the dendritic marker MAP2, Huang reported. In contrast, mice expressing a shorter, 241-amino acid protein that lacks the lipid binding domain seem normal, indicating that lipid interaction may be essential for the truncated ApoE’s neurotoxicity.

The physiological relevance of this work has been questioned because of lack of evidence that neurons normally express ApoE, but Qin Xu in Huang’s group showed that ApoE can be expressed in CNS neurons under stress (SfN abstract 442.6). Using immunostaining and in situ hybridization, Xu showed that ApoE is expressed in CA1 hippocampal neurons that are subjected to excitotoxic injury using kainic acid. Xu has produced chimeric mice that have green fluorescent protein expressed under the endogenous ApoE promoter and hopes to use these animals to probe, in real-time, changes in expression of the lipoprotein.—Tom Fagan.

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San Diego: AβPP—Can the Tail Wag the Dog?

23 Nov 2004

Since the discovery that one peptide, amyloid-β, is the major constituent of amyloid plaques, there has been a tremendous focus on the N-terminal end of amyloid-β precursor protein (AβPP). But as emphasized at the 34th annual meeting of the Society for Neuroscience in San Diego last month, the C-terminal end of AβPP has its claim to fame, too.

Work from the labs of Dale Bredesen and Eddie Koo has previously shown that a single caspase-mediated cleavage releases the last 31 amino acids from the C-terminus of AβPP. This C31 peptide causes apoptosis in cultured cells and can be found in brain samples from AD patients but not in samples from control brains (see ARF related news story). But does it have a role in vivo?

Apparently, it does. Continuing the Californian collaboration, Veronica Galvan from the Bredesen lab at the Buck Institute for Age Research, Novato, and Brock Schroeder from Eddie Koo’s lab at University of California San Diego reported that in transgenic animal models of AD, lack of this cleavage site in the C-terminal of AβPP has profound effects.

To test how mutation of this cleavage site may affect the progression of AD-like pathology, Galvan substituted an alanine for asparagine at position 664 of the human AβPP gene in PDAPP mice. (Asparagine 664 is where the C31 cleavage occurs; see SfN abstract 488.4.) PDAPP mice express human AβPP harboring mutations that cause early onset AD in humans (phenylalanine for a valine at position 617, asparagine for lysine at 670, and leucine for methionine at position 671). These mice develop Aβ plaques and suffer synaptic and neuronal losses, and develop memory deficits as they age. When Galvan examined mice expressing the alanine664 AβPP, she found that they produced about the same quantity of Aβ and about the same number of plaques as asparagine644 PDAPP animals. However, synaptic and hippocampal changes were a different story. Galvan found that hippocampal volume—normally reduced by about one-third in PDAPP transgenics—was maintained in mice devoid of C31. The loss of synaptic densities (up to 50 percent) that normally occurs as the PDAPP mice age was also prevented, as judged by immunoreactivity of the synaptic marker, synaptophysin. The results suggest that the neurotoxicity that accompanies accumulation of Aβ plaques in these mice can be prevented if the C31 cleavage is abolished.

Continuing with this theme, Schroeder showed that synapses are indeed protected in PDAPP mice that can’t produce C31 (see SfN abstract 488.5). In these animals, not only is loss of the synaptic protein synaptophysin attenuated, but also extracellular field potentials recorded from hippocampal neurons are maintained instead of reduced as they are in standard PDAPP transgenics (by about 50 percent).

All told, the physiological data suggest a major role for the C-terminal cleavage of APP in the pathology of AD. So might it contribute to the most worrisome syndrome of AD, cognitive impairment? To test this, Schroeder evaluated PDAPP mice in the Morris water maze, which measures spatial learning and memory. The animals lacking C31 failed to show the loss of spatial learning and memory that PDAPP mice normally exhibit as they grow older.

So how may the C-terminal cleavage of APP contribute to pathology in these animal models? Galvan suggested two possibilities: Cleavage at asp644 not only yields C31, which is toxic in vitro, but also lops off endocytic signals and binding sites for the transcriptional associated factors Fe65 and X11, leaving the AβPP intracellular domain (AICD) with no means to attract these partners. This C-terminal cleavage may, therefore, interrupt both endocytosis and AICD signal transduction to the nucleus. If this turns out to be true, then facilitating AICD signaling could be a novel therapeutic approach to treating AD.

So can we now forget about the head of AβPP, the Aβ? That is unlikely, given the amount of evidence linking it to neurotoxicity, and also recent observations showing that Aβ may bind to AβPP, induce its dimerization, and favor the C-terminal cleavage of the precursor (see Lu et al., 2003 and also related news), and that C31 alone is insufficient for toxicity—full-length AβPP is needed as a co-conspirator (see Lu et al., 2003). So while the observed lack of AD-like pathology in PDAPP-ala664 supports the involvement of both the C- and N-terminal ends of AβPP, perhaps the tail does wag the dog a little.—Tom Fagan.

References

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Paper Citations

Lu DC, Shaked GM, Masliah E, Bredesen DE, Koo EH. Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes. Ann Neurol. 2003 Dec;54(6):781-9. PubMed.
Lu DC, Soriano S, Bredesen DE, Koo EH. Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity. J Neurochem. 2003 Nov;87(3):733-41. PubMed.

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San Diego: AβPP—Can the Tail Wag the Dog?

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