CONFERENCE COVERAGE SERIES
World Alzheimer Conference 2000
Washington, D.C.
09 – 18 July 2000
World Alzheimer Conference 2000: De la Monte Receives Alzheimer Medal
The editors of the Journal of Alzheimer Disease announced today that Suzanne de la Monte was the recipient of its Alzheimer Medal for best article published in the past year. De la Monte was lauded for her article, "Cerebrovascular pathology contributes to the heterogeneity of Alzheimer's disease." Details on the award and Dr. de la Monte's work can be seen at http://www.j-alz.com/award.html
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World Alzheimer Conference 2000: Symposium on ApoE
Amyloid plaques are still the main attraction, and tangles have certainly not left the stage, but there is still a lot of interest at this meeting (off-Broadway, so to speak) in other potential players in AD pathology. An example is ApoE, widely recognized as the most common risk factor for the disease. A panel of experts provided their views on the potential role of this protein in AD pathology. Although there was a disproportionate amount of previously published work presented by most speakers, there were enough new results (especially from Weisgraber) to maintain the interest of the relatively small audience. Ever since the genetic association between the ApoE4 isoform and AD risk was discovered by Allen Roses and colleagues, a number of laboratories have been developing and testing hypotheses as to what this lipid-transport protein might be doing. As pointed out by Judes Poirier, one of the presenters, these hypotheses fall into two broad classes: ApoE (especially E3) is good or ApoE (especially E4) is bad. The majority of hypotheses are based on the notion that E4 is simply not as good as E3 in performing some vital function.
Whether or not E4 is bad or just ineffective, Weisgraber described strategies for studying its effects in a transgenic mouse model and for making it more like E3. This group has previously found that "domain interactions" between Arg-61 and Glu-255 are important in making E4 behave differently from E2 or E3. The novel mouse is one in which mouse ApoE has been "humanized" by replacing the normal threonine at position 61 with arginine. (Of several species examined, human ApoE is the only one with arginine at this position.) The ApoE from this humanized mouse shows preferential association with VLDL, as does human ApoE4. The humanized gene is under the control of the natural mouse enhancers and tissue-specific elements and there is no endogenous mouse ApoE expressed. These mice will be studied as potential models for biological effects of ApoE4. Weisgraber also described a drug-screening strategy to find compounds that will specifically interfere with the domain interaction present in E4. A molecular modeling approach (the DOCK program) was used to assess how well putative ligands fit into the site that mediates interaction between the critical residues in E4. 65 compounds showed inhibition of the VLDL-binding preference of E4 when tested in an emulsion assay. Thirteen of these compounds reduced binding activity of E4 to that normally obtained with E3 but had no effect on E3 binding. The goal is to identify compounds that make E4 behave more like E3 as a means of treating the ApoE-related pathology of AD.
David Holtzman followed with a review of mostly published studies involving several transgenic mice in which APP is overexpressed in the presence or absence of different ApoE backgrounds. The absence of enodgenous ApoE results in less Aß deposition in brain, with almost no fibrillar Aß and no dystrophic neurites at 12 and 15 months of age. In the presence of ApoE, however, abundant thioflavin S-positive plaques with dystrophic neurites are present. Lack of ApoE also reduces vascular amyloid deposition. When human ApoE is expressed in APP mice (without mouse ApoE), there is initial suppression of amyloid deposits. However, E4 mice show 10-fold greater Aß deposits and neuritic plaques than E3 mice at 15 months of age. This isoform-specific effect is pronounced at 21 months when the E4 mice show significant neuritic dystrophy. The hypothesis is that ApoE plays a critical role in conversion of Aß to fibrillar (toxic) forms and that E4 may be doing this more than E3. (Surprisingly, Holtzman did not mention the possibility that ApoE may itself be a source of neurotoxic activity.) He also presented more recent data on the levels of Aß in CSF collected from the mouse cisterna magna (a heroic task!). There is a decline in CSF Aß in the E4 mouse over time, perhaps due to deposition or clearance and Aß levels are correlated in CSF and plasma, suggesting that clearance into plasma may be occurring.
Poirier provided an overview of studies based on the assumption that ApoE normally plays a positive role that needs to be enhanced in order to modify the pathology of the disease. He noted that others have shown that plasma ApoE is lowest in E4 homozygotes. Poirier's group has shown the same result in AD patients with similar trends for hippocampal ApoE levels (lowest in 4/4 samples). Low throughput screening was used to identify compounds that will increase ApoE production in rat astrocytes on the assumption that boosting ApoE will be of benefit in treating AD. A wide variety of agents were identified, one of which, Probucol, was found to be especially potent in vitro and was also found to cause increased ApoE in mouse brain in vivo. An initial clinical trial has been carried out with Probucol in AD patients in which ApoE levels in lumbar CSF showed an average increase of 20% but the increase was greatest in subjects lacking the E4 allele. Interestingly, the cognitive status of these patients appeared to stabilize and there was a strong positive correlation between cognitive performance and CSF ApoE.
Bales returned to transgenic mice, reviewing mostly published data demonstrating that the absence of ApoE results in no thioflavin S plaques and no neuritic dystrophy in transgenic amyloid mice. Also, there is a different pattern of amyloid staining in the absence of ApoE. When ApoE is present, it appears in all the thioflavin S-positive plaques (as it does in AD plaques). Replacing mouse ApoE with human apoE demonstrates that E4 mice develop thioflavin S-positive plaques, but there are none in E2 or E3 mice. She also reported on APP mice that were fed a fat-enriched diet for 12 weeks. This resulted in more Aß staining and more thioflavin S-positive plaques.
Mucke also covered transgenic territory in the context of understanding gender- and age-dependent effects of ApoE on disease risk. Women are more affected than men by the presence of E4, although this effect is less prominent at early and later ages. In APP mice, dystrophic neurites are associated with plaques and there is an age-dependent decline in synaptophysin staining [SYN-IR], which negatively correlates with Aß42 levels. Hippocampal slices from these mice exhibit deficits in basal synaptic transmission but these effects are independent of plaque load. One possible explanation is that plaque formation is separate from degenerative effects of Aß. When combined with ApoE transgenic mice (under control of GFAP or NSE), behavioral deficits emerge in the E4 mice but not the E3 mice. In fact, E4 may exert a dominant negative effect under some conditions, perhaps through effects on Aß, but there also may be differences in the ApoE isoforms on neuronal protection. He also reported on gene chip analyses to look for changes in hippocampal gene expression in these mice. 618 of 13,033 screened genes showed changes. It remains to be determined which ones will be informative.
Smith rounded out the symposium with a return to the gender gap as well as evaluating the contribution of trauma (head injury is a risk factor for AD). Mice in which ApoE expression is driven by the GFAP promoter were studied. Estrogen results in induction of ApoE in the brain, but with regional variations. Ovariectomy results in increased mortality in APP mice, an effect that is apparently not due to increased Aß. Using a fluid percussion head injury model, induction of ApoE and GFAP were found. He also examined the contribution of environmental or genetic variations to phenotypic results obtained with transgenic lines. C57BL/6 and FVB/N mice were studied with and without endogenous ApoE. The FVB/N mice are good breeders but harbor the rd mutation that causes blindness. Using an olfactory-cued eight-arm radial maze (does not require vision), all six-week-old mice showed learning over five days. However, at six months, only the C57 ApoE-/- mice show deficits, which are not due to increased corticosterone. Thus, both age-dependent and genetic strain-dependent effects on cogniitve behavior exist and need to be considered when evaluating transgenic models.
If there was a common message in this symposium, it was hard to decipher. ApoE clearly has an effect on amyloid deposition and there are interesting isoform-specific effects of ApoE on transgenic mouse phenotypes. However, whether ApoE ever gets the chance to share the limelight with amyloid or tau is unknown and will likely depend on a unifying hypothesis that still remains to be established for ApoE's role.—Keith Crutcher
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World Alzheimer Conference 2000: Symposium on Therapeutic Approaches
One of the first symposia of the conference was dedicated to an overview of the current and future treatments for AD. Six presenters gave their perspectives on many treatment issues ranging from management of behavioral disturbances to new drug targets under development. On the whole there was a tone of optimism tinged with the reality of the limitations of current therapies.
Jeffrey Cummings considered the topic of behavioral management, noting that there are no current FDA-approved therapies for the treatment of AD-related behavioral disturbances. However, a number of medications used for the treatment of psychotic symptoms have been shown to be effective in AD, e.g., olanzapine and risperidone. A more common behavioral problem is agitation (up to 70 percent of cases) and increases with severity of disease. Haloperidol does not appear to be very effective, but risperidone does have efficacy. Major depression is infrequent but symptoms are present in 40 percent of patients. A number of common antidepressants appear to be effective, e.g., citalopram but there also appears to be a placebo response in many patients. Aricept and Exelon, which are used to treat mnemonic deficits, may also have signficant psychotropic properties.
Rachelle Doody provided an overview of cholinesterase inhibitors, several of which are already approved or currently in development. All appear to enhance cognition in at least certain patients. Side effects are mainly gastrointestinal complications. Dr. Doody reviewed the evidence for efficacy of these agents. Maximum effects depend on the measures used. None appeaer to show more than a 10 percent improvement on a variety of scores when mean group differences are used. However, she noted that efficacy can be quite dramatic in some patients, including those with severe dementia. There is little information available on efficacy for MCI patients (mild cognitively impaired) and for profound AD or other types of dementia. She noted that discontinuation of treatment carries a risk of not regaining baseline status after treatment is resumed but that benefits are of long duration if treatment is not interrupted. She also pointed out that the available evidence does not appear to converge on the original cholinergic hypothesis, which is now considered to be incomplete at best.
Kenneth Rockwood provided an overview of noncholinergic therapies, including recent disappointments of several drugs that have failed in initial trials, e.g., estrogen, prednisone, celecoxib, propentofylline. Specific disease-modifying drugs are some time in the future. In the meantime, there is a focus on prevention, combination therapies and some adjustment of expectations of current treatments. New strategies may include reducing Aβ production, reducing tangles, promoting repair, slowing neuronal death, and mitigating inciting events. Promising leads may come from anti-hypertensives. Another point Rockwood made was that some reports of ineffectiveness may be due to off-setting changes that are reported as no change, e.g., memory may not improve but other symptoms may. Ultimately combination therapies will probably be required.
Steve DeKosky gave an overview of anti-amyloid strategies noting that some drugs may have a nonspecific impact on amyloid. The goal is to develop agents that will act more specifically, such as β-secretase inhibitors, gamma secretase inhibitors, α-secretase enhancers, or agents that accelerate clearance of Aβ, or block amyloid aggregation. Since it is still not clear what the normal function of APP is, these strategies may involve undesired effects. Triggers for Aβ production include normal aging, APP mutations, PS-1 and PS-2, and altered second messenger systems. Other novel approaches include immunization to clear amyloid (through macrophage activity) or to prevent further deposition.
Steve Paul reviewed the evidence collected from transgenic mice on the role of ApoE. ApoE has been suggested to work through neurotrophic effects (E4 being less effective), as an antioxidant, or through effects on tau binding or amyloid deposition. The latter is supported by epidemiological data as well as the results from transgenic mice (APP V717F +/+ x apoE -/-) in which amyloid deposition is largely prevented by the lack of ApoE. ApoE appears to be involved in promoting fibrillization of Aβ as well as the formation of neuritic plaques. There is also reduced astrogliosis in the absence of ApoE.
Collectively the results are interpreted as indicating that ApoE contributes to disease risk by enhancing amyloid deposition. Dr. Paul also reported recent data in which infusion of LPS into the lateral ventricle of the APP mice resulted in three and fourfold acceleration of amyloid deposition in the presence of ApoE whereas the ApoE -/- mice still show no plaques. Current work is focused on strategies to reduce ApoE synthesis through manipulations of intracellular signaling pathways that implicate NFkB.
The symposium was rounded out by a review of cholinesterase inhibitors by Dr. Giacobini. He noted that the original assumptions underlying the cholinergic hypothesis do not appear to hold up in terms of understanding the effects of these inhibitors. He noted that there may be some effects that are not limited to the anticholinesterase activity of these agents in light of the fact that AChE also has binding sites for other molecules implicated in AD, such as amyloid and the fact that there is some evidence for cholinergic regulation of APP synthesis, possibly mediated through muscarinic receptors. This emphasizes the complexity of the establishing an effective treatment. All of the presenters agreed on the difficulty of developing a single treatment based on a single mechanism of disease pathology. The task is not easy but the ground that has been gained so far suggests that the next few years may well provide encouraging news on the eventual development of drugs that not only reduce negative symptoms but intervene in the insidious course of the disease. The challenge remains.—Keith Crutcher
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World Alzheimer Conference 2000: Drosophilae in the Emulsifier
The centrality of the amyloid hypothesis for AD pathology is evident at this conference not only by the sheer number of papers and posters devoted to it (708 out of a total of 1279, as opposed to 163 for apoE and 152 for presenilin, for example), but also by the tremendous excitement surrounding the announcement by Schenk that treatments based on immunization to this protein are coming along quite nicely. But is all well in amyloidville? The occasional fly keeps settling on the body of evidence that has accumulated regarding the premise that this protein is truly the cause of the neuronal death and destruction in the AD brain. A rather large fly landed today in the form of a poster from Glenda Bishop and Stephen Robinson (Abstract 1027) concluding that intracerebral injections of Aß in the rat do not give rise to obvious toxicity. In fact, they reported that such injections protected against injury caused by ferric ammonium citrate injections. (Well, they are from down under, maybe things work backwards in the southern hemisphere?) This is not the first time that amyloid has not lived up to its reputation as neuron-slayer, but most investigators who fail to find toxicity usually keep it to themselves. A second fly could be found only two poster aisles away by a group from Elan (Abstract 1188) claiming that neither the monomeric nor the fibrillar form of amyloid (1-40) is toxic. However, they did find toxicity if they pretreated the cells with the fibrillar form and then followed with the monomeric form. The conclusion being that ongoing fibrillization is necessary to observe toxicity. This proposed mechanism may in fact be the explanation for the not uncommon failure to observe amyloid toxicity in vitro and in vivo. It may also be consistent with the observation that only fibrillar plaques in transgenic mice appear to be associated with neuritic pathology. If ongoing fibril formation is necessary to observe toxicity, any means of interfering with this process could theoretically be an effective means of countering amyloid toxicity, including the vaccine approach.—Keith Crutcher
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World Alzheimer Conference 2000: Neuroinflammation in AD
Reported by Walter Lukiw. www.elsevier.com/locate/niad2000
Classical and Alternative Pathways-Neil Cooper.
It has been a century since Alzheimer and Bielchowsky reported senile plaques (SP) in AD brain, but it was only relatively recently appreciated that SPs are embraced by reactive astrocytes and microglia. This combined lesion complex of SP and reactive microglia acts as a potent activator of the complement system-a complex multicomponent biological effector with ~30 components, some circulatory, some on neuronal and/or glial membranes. Aβ peptides and SP activate complement by binding C1q, generating proinflammatory (PI) C5b-C9 membrane attack complexes. Notably, transgenic βAPP overexpressors when crossed with C3 mutants dramatically increase (processed) Aβ deposits.
Complement Regulation-Piet Eikelenboom
Complement C1 and C3 have been implicated in maintaining the solubility of Aβ. As far back as early 1982, Eikelenboom's group showed that the main source of complement was neurons and not glia. It is the microglia that generate PI cytokines such as IL-1β which induce neurons to make βAPP and complement; the neuron is just not an innocent bystander in PI pathways in AD brain. Therapeutic intervention to augment complement activation may be best aimed at serine proteases C1r/s (C1 subcomponents) which would block C1 activation before C3, the major effector protein of the complement cascade, is activated.
The Role of Complement Anaphylatoxin C5a in Neurodegeneration-Implications for AD-Giulio Pasinetti.
Mice deficient in C5 complement are more susceptible to hippocampal excitotoxic lesions. C5-derived anaphylatoxin C5a may protect against excitotoxicity both in vitro and in vivo. Mechanisms of C5a-mediated neuroprotection include MAP kinase activation. In transgenic C5a receptor knockout mouse (C5aR-KO), alteration of C5aR alters calcium/calmodulin kinase signal transduction detrimental to normal brain cell function. In AD, an increase in cyclooxygenase-2 (COX-2) expression has been reported at CDR 0.5-1.0, right around the time of the first signs of Aβ plaque density increases. COX-2 presence and elevations are a key marker for AD onset.
α1-ACT, α2-MAC, Serum Amyloid P-Ikuo Tooyama.
Acute phase reactants are a heterogeneous collection of proteins upregulated in blood plasma when PI responses occur. These are chiefly α1- antichymotrypsin (ACT), α2 macroglobulin (α2 MAC) and serum amyloid P (SAP). α1-ACT is a serine protease inhibitor involved in PI, strongly binds to Aβ and stimulates its polymerization (bad stuff!), and also contributes to the resistance to proteolytic degradation. α2 MAC is also a potent proteinase inhibitor-also binds to Aβ and prevents its degradation (more bad stuff!!). In AD brain, α2-MAC may also act as a chaperone for Aβ and prevent Aβ clearance. SAP, a serum plasma protein that is present in all SP deposits, binds complement proteins C4 and C1. The general upregulation of such acute phase proteins cumulatively is bad news for AD brain.
Fibrinolysis/coagulation systems-Haruhiko Akiyama.
AD lesions inducing PI cascades resemble chronic inflammation in the periphery. Just as it functions in the periphery, thrombin in the brain causes rapid retraction of neuronal and astrocyte processes. Fibrinolysis is involved in neurite outgrowth, neuronal migration, neuronal plasticity and excitotoxicity. Secreted APP (sAPP) is identical to protease nexin-2 (down-regulated in AD). Nexin-2 inhibits coagulation, indicating a physiological role of the intrinsic coagulation pathway in AD brain. Coagulative process increased in AD brain-it can be found in all mature SP.
Cyclooxygenase-1, Cyclooxygenase-2-Kerry O'Banion
McGeer et al. (1990) were some of the first to point out that AD prevalence in patients with rheumatoid arthritis (taking NSAIDs ) was 6-12 times less frequent then expected. Rogers et al. (1993) first showed AD patients treated with indomethacin showed reduced progression of AD. Since COX's were one of the known targets for NSAID action, these data both suggested the probable involvment of inducible cyclooxygenases (COX-2) enzymes in AD brain. A key marker for AD brain is increased IL-1β, verified by many groups. Upregulation of IL-1β is a super potent inducer of COX-2 gene expression. Aβ doesn't increase COX-2 by itself but IL-1β and Aβ show synergistic induction of COX-2 in AD brain. COX-2, as the key enzyme in PI mediator activation is elevated in AD brain and is the target of several anti-PI pharamacological studies. These include the specific COX-2 inhibitors NS-398 (Taisho), Celcoxib (Searle) and Rofecoxib (Merck). Large trials for AD are currently under way and preliminary results are promising.
IL-1/S100β-W.S.T. Griffin, U. Arkansas, USA.
Griffin and coworkers have shown that IL-1β: (1) is overexpressed by microglia in AD brain; (2) is overexpressed in areas with the most Aβ deposition; (3) induces excessive synthesis and processing of βAPP in neurons; (4) overexpression correlates with the transition from fibrous Aβ to condensed amyloid senile plaques (SP) and (5) strongly activates astrocytes. IL-1β converting enzyme (ICE), also known as caspase 1, the enzyme that converts pro-IL-1β to mature functional IL-1β, is also up regulated in AD. Overexpression of ICE contributes to DNA damage observed in neurons adjacent to SP. IL-1 may have a more direct function in cholinergic dysfunction. Overexpression of IL-1 induces expression of acetylcholinesterase both in vitro and in vivo. Finally, specific polymorphisms in several IL-1 genes increase risk for AD.
IL-6-Joachim Bauer.
IL-6 is a pleotrophic cytokine that mediates immune responses and PI reactions affecting cell growth and differentiation. Primary IL-6 synthesis is in human brain microglia and astrocytes. It is a major pyrogen and increases vascular permeability and lymphocyte activation-all of which have been shown to be elevated in AD brain. IL-6 RNA message and protein increases rapidly as little as 30 minutes after prostaglandin E2 (PGE2) treatment of astrocytes. Prostainoids (COX-2 end products) such as PGE2 induce astrocytic production of IL-6. NSAIDs inhibit COX-2 but do not overtly inhibit IL-6 synthesis in astrocyte cultures. More specific inhibitors of IL-6 are one important and attractive targets for AD therapy in the future.
TGFβ-Tony Wyss.
Transforming growth factor β's (TGFβ's) are multifunctional polypeptide growth factors which play prominent roles in tissue development homeostasis and repair and are expressed in all brain cells-neurons, astrocytes, and microglia. Just like for other cytokines, for TGFβ, too much of a good thing is a bad thing. TGFβ is also widely found in SP cores, in CSF and serum of AD cases. TGF can be a strong promoter of PI mechanisms. Cross-breeding of TGF transgenic mice with hAPP mice promote deposition of Aβ in cerebral blood vessels rather than in plaques. Some subforms of TGFβ (β1 and β2) are shown to protect against neuronal injury by inhibition of apoptosis through induction of Bcl-2 and stabilization of intracellular calcium.
Summary-Nancy Ruddle, Yale University. 1. Most if not all PI mediators are increased in AD brain and associated with classical neuropathological structures such as the SP.
2. Cyclic pattern of the PI response in AD brain is positively reinforcing, so stopping anywhere around the cycle might be expected to stop the feed-forward mechanism (ideally).
3. Don't blame everything on the glia/astroctyes. Neurons can do a lot of damage to themselves and to glia.
8 July 2000. Microglia, or "The Natural and Un-Natural History of Brain Microglia."-Mark Emmerling
Microglia are the resident macrophages of the CNS, make up ~10% of total CNS cells and perform many functions of peripheral macrophages including migration, phagocytosis, and production of immune molecules like cytokines and chemokines. Microglia may be a good pathological marker for AD due to the following reasons: (1) Natural aging shows an elevated microglial activation response which in AD is exacerbated (see Streit, Progress in Neurobiol 1999 57:563-581). The difference between "resting" microglia and "activated" microglia is phenomenal. This can raise microglial IL-1β RNA message production by a factor of 1,000 to 10,000 (!); (2) Aβ, and deposition into SP are the key pathological markers for microglial activation. Aβ induces membrane perturbation through membrane-binding sites for Aβ (the serpin enzyme complex receptor), C3b, C5 activation products, heparin sulfate proteoglycans, LDL receptor related proteins, RAGE, Aβ binding alcohol dehydrogenase (ABAD)-all strongly PI. Interaction of Aβ with microglia ends up generating more IL-1α, IL-1β, etc. Microglial targeted treatments should focus on NF-κB antagonists and PPAR c agonists (e.g., propentofyline, a potent anti-PI compound).
Astrocytes-Robert Mrak.
Activated astrocyte products are invariable components of SP in AD and suggest an instigating or proliferative role. Astrocyte products in AD include trophic molecules, adhesion molecules, cytokine S100β-all PI associated proteins. S100β normally promotes neurite outgrowth, maintains free calcium levels and when dysregulated induces neuronal βAPP gene expression, IL-6 and NOS activities. A strong correlation exists between S100β-expressing astrocyte density and SP number. IL-1β, shown by many reports to be elevated in AD induces S100β, αACT, ApoE and C3 as part of an agressive "cytokine cycle" in AD brain.
Neurons-Steven Barger.
βAPP and Aβ are the key PI markers for AD. Their lab sees an induction of the inducible prostanoid generating COX-2 RNA and protein (isozyme) in global ischemia models. They also observe a dramatic induction of the proinflammatory transcription factors (TF) AP1 and NF-κB, but not of TF Oct1 or SP1 in iscehmia. Glutamate increases NF-κB-DNA binding only in mixed neuron-glia cultures suggesting neuron-glia crosstalk in initiating PI pathways. A neuron specific NF-κB in rat cultures they called NK-BF for neuronal K B factor. NK-BF may be rat-specific and show only rat-specific responses in PI signaling. sAPP, the "good" amyloid derived from APP is generally thought of as neuroprotective, but can induce IL-1 after further "abnormal" sAPP processing in PI brain cells. The sAPP N-terminus appears to be PI, while the sAPP COOH terminus appears to be neurotrophic (impications for α-, β-, or c- and other secretase activities in contributing to PI in AD brain).
Aβ Binding Proteins-Scott Webster.
Aβ binding proteins (1) modulate formation of Aβ and SP; (2) affect induction of Aβ mediated PI pathways and neurotoxicity; (3) influence Aβ clearance mechanisms such as the susceptibility of Aβ to proteolysis; (4) influence Aβ as a target for phagocytosis; (5) modify Aβ-mediated stimulation of PI-mediator production; and (6) alters βAPP gene expression pathways. C1q, SAP and ACT are amyloidogenic. ApoE and α2 MAC are anti-amyloidogenic.
Oxidative Stress-Douglas Walker.
Free radicals are constantly produced by cells as the result of oxidative metabolism. The brain has a high metabolic rate so there are lots of reactive oxygen species (ROS) to deal with. When clearance of ROS is compromised, oxidative stress is increased as appears to be the case in AD. Aβ peptides can prime ROS production in rat microglia (Klegeris et al., 1997). ROS can also induce phagocytosis of glia and IL-1β release. Aβ at 10 uM induces RAGE (receptor for advanced glycation end-products). Markers of oxidative stress in AD brains include elevated advanced glycation endroducts, malondialdehydes, elevated 4-OH nonenol (indicated hyperoxidized lipids), elevated carbonyl (oxidized proteins), nitrotyrosine modified proteins, etc. These changes are also seen in transgenic mice that are βAPP overexpressors. Antioxidant therapy may be beneficial in dealing with ROS in AD brain.
RAGE and the Macrophage Scavenger Receptor-David Stern.
RAGE is a multiligand member of the IG superfamily of cell surface molecules and contributes to cell oxidative stress accompanying amyloidosis. Amyloid interaction with cell surface RAGE promotes accumulation of Aβ fibrillation. RAGE is down-regulated during human brain development and is up-regulated in pathological states just like basal levels of COX-2. The RAGE promoter contains multiple NF-κB-DNA binding sites and the Tfs cINF and NF-IL6 are important regulators of RAGE transcription. RAGE activation increases production of macrophage colony stimulating factor (MCSF) and IL-6-anti-RAGE antibodies block this induction. When neurons bind Aβ they also produce MCSF. MCSF activates microglia to produce more IL-1, and the PI cycle increases by a feed forward mechanism. Inhibiting key components of the RAGE pathways may be useful therapeutically.
Summary-Patrick McGeer.
"The lesions of AD are characterized by the presence of a host of PI molecules." Chronic inflammation is involved in AD, heart attack (MI), and stroke, which take three of four people prematurely. NSAIDs reduce the risk of MI 25%-85%, AD 50%-75%, stroke, colon and breast cancer, and cataract 25%-50%. McGeer referred to the flak he and Joe Rogers took 15 years ago when they first used the term "neuroinflammation"-then considered an oxymoron-which is now of mainstream AD interest. The concepts of PI processes in human brain were reviewed historically, from Metchnikoff (1843-1916) who first described phagocytes, a local PI process and Hortega, who first suggested that brain microglia gathered as phagocytes during brain injury, to Ralf von Furth who described microglial activation in the brain (and could never get funded) to Jules Bordet (1870-1961) who described brain complement, rubor (cytokine induction) and also said inflammation in local. In summary: AD is a mixture of autoimmune and autotoxic processes. Inflammatory outcomes are (1) healing, (2) death, or (3) chronic inflammation, which is the stalemate situation observed in AD. Note that antiinflammatory compounds (aspirin) are the most prevalent class of drugs ever developed by man. NFT involvement in the induction of PI cascades was briefly disussed. Both senile plaques and NFT are PI inducers. To date, NSAIDs are the preferred alleviator molecules for quenching PI pathways.
Future Directions
Choosing the right drug and the right time for AD neuroinflammation therapy-John Breitner.
The AD anti-inflammatory prevention trial (ADAPT) is now funded and will assess efficacy of the conventional COX1/COX2 inhibitor naproxen and celecoxib (selective COX2 inhibitor) for primary prevention of age-related and AD cognitive decline. Trials of hormone replacement therapy and histamine H2 blockers are attractive (no major side effects). The aim is to prevent onset of AD by retarding the progression of the disease in its latent stages. The compounds lose effectiveness in later stages. Short trials to date have been to date discouraging.
Amyloid Vaccination and Its Neuroimmunologic Implications-Dale Schenk.
Aβ immunization of PDAPP mice works remarkably well in preventing Aβ deposits from forming and in reducing progression of plaque deposits and neuropathology in older animals. Therefore, a directed immune response against Aβ can be beneficial in reducing AD-like pathology.
Neuroimmunology of Aging-Caleb Finch.
Normal aging involves subsets of the same PI phenomenon as in AD, and AD is an "accumulated aging" response. In normal aging, there are increased deposits of various tissue amyloids, but they differ from Aβ in the brain. AD is part of a broad "gero-inflammatory" response fueled by the oxidative processes associated with aging.
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World Alzheimer Conference 2000: Huang Poster
The potential contribution of ApoE to AD pathology was cast in a novel light in data presented as a poster today by Huang et al. They were studying the fate of ApoE when expressed by transient transfection in Neuro-2a cells. The ApoE was tagged with green fluorescent protein (GFP) to allow for visualization of its intracellular fate. Previous studies from this group had shown that full-length ApoE exhibits isoform-dependent differences in the cellular distribution of the protein. In the present study, ApoE was truncated at its carboxy terminal end (residues 272-299) to see if this would affect its intracellular fate. Surprisingly, the truncated ApoE was found to produced tangle-like structures in the cells, with the ApoE4 isoform being much more effective than the apoE3 isoform. The tangle-like intracellular structures stained with an anti-tau antibody (RT97) as well as with antibodies to neurofilament heavy chain. They appeared to be composed of dense bundles of filaments at the electron microscopic level. This aberrant intracellular formation apparently was toxic, because the cells eventually died (MTT assay). Similar isoform-specific effects were obtained if the truncated GFP-ApoE was added exogenously. Although the mechanism is unknown, the demonstration of isoform-specific effects of ApoE on cytoskeletal structures is consistent with a possible role for this protein in tangle formation in AD. Huang says that transgenic mice have been developed using similar constructs and are awaiting analysis.—Keith Crutcher
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World Alzheimer Conference 2000: Maybe Plaques Kill Neurons after All
Although there are many reports of neurotoxic effects of amyloid when applied to neurons in culture, the evidence for toxicity in vivo has been less compelling. Of particular surprise has been the finding that most transgenic mice in which abundant plaques form, show little evidence of neuronal loss. Bradley Hyman’s group has carried out much of these analyses. Is it possible that a subpopulation of plaques affect neurons in their immediate vicinity? This was the question addressed by Hyman’s group using a statistical model based on methods adapted from solid state physics. Transgenic mice (Tg2576 x PS1 M146L) were studied using double-immunofluorescence labeling of plaques and neurons. No global loss of neurons was found. However, when local analysis was carried out, local neuronal density was found to depend on the size and density of Aß deposits. For most plaques there was no effect on regional neuronal density. However, when subdivided on size and intensity of Aß staining, a subpopulation of plaques (about 2% of all plaques) was found to be associated with local decreases in neuronal density. These plaques stain with Thioflavin S, presumably due to dense fibrillar deposits of amyloid. In order to determine whether the decreased neuronal density was due to displacement of cells or neuronal loss, computer modeling was used. The results suggest that the plaques are not simplying "pushing" neurons aside but are truly causing cell loss. The reduced density is limited to the immediate region of the plaque, indicating that there is not a diffusible toxic signal emanating from the plaques. Whether these effects are indicative of a similar situation in human AD remains to be determined. However, the fact that some plaques in transgenic mice appear to cause local loss of neurons renews hope that these models will yet provide critical clues to understanding the pathological cascade in the Alzheimer brain.—Keith Crutcher
References: Hyman B. Assessing neuronal loss in transgenic models. World Alzheimer Congress 2000 Abstract 325.
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World Alzheimer Conference 2000: Hot Topics in AD
Several late-breaking, dramatic results were presented at this final symposium of the meeting. Michael Hutton and colleagues from Mayo Laboratory, Jacksonville, which only a few days earlier had first publicly presented data showing tangles and neurodegeneration in an FTDP-17 tau (P301L) transgenic mouse, revealed that double transgenics expressing the swedish APP mutation and P301L tau also had NFTs and neuropathology, as well as Aβ plaques.
Yong Shen of Sun Health Research Institute described a successful method to culture neurons harvested postmortem from sporadic AD brain. He noted that the AD neurons developed fewer processes but, curiously, lived longer than neurons cultured from normal adult brains. It will be of great interest to examine the molecular and cell biology of the AD neuronal cultures, and they offer an intriguing possibility for drug assays.
David Morgan from University of South Florida presented data showing that Aβ vaccination blocked cognitive deficits in PS/APP double transgenic mice. Using a novel spatial learning task-a six-arm radial water maze-the USF investigators found that 12-month-old and 15-month-old mice that had received a course of vaccinations from four months of age onward performed as well as control animals, learning to navigate the maze with few errors. In contrast, age-matched nonimmunized mice were unable to learn the task and performed at chance. What’s more, 15-month-old mice that received a shorter course of vaccinations also improved to almost the same performance level as the controls. Fascinatingly, the 15-month-olds that had short-term vaccination had almost no reduction in Aβ burden compared to nonimmunized mice, suggesting that the plaques per se were not responsible for the observed cognitive deficits. One possible explanation is that immunization is leading to clearance of solube Aβ, which is causing the impaired function.
One hypothesis to emerge from the Aβ vaccination studies is that plaques may accumulate in Alzheimer’s patients because they are unable to clear Aβ as effectively as people who don’t get Alzheimer’s. Yangsheng Du presented data to support this notion, showing that CSF from Alzheimer's patients had a 30% reduction in levels of Aβ antibodies.
Miguel Pappolla presented data showing that high doses of melatonin produced dramatic drops in Aβ in the Tg2576 ("Hsiao") mouse. The mice were given around 1.5 mg per day of melatonin from age four months onward, and sacrificed at 15 months. The melatonin also produced "significant improvement" in performance on the Morris water maze, Pappolla reported.
In the final talk, Barry Reisberg presented preliminary results from the first U.S. clinical trial of memantine, a drug that targets the NMDA receptor system and which has been marketed in Germany. In the study, 126 patients with moderately severe to severe Alzheimer’s received 10 mg doses of memantine twice a day, while 126 matched controls received placebo. After six months, the treated patients were found to decline more slowly than the placebo group on a number of standard measures (which I was unable to note down because the data flashed by so quickly). Thus memantine may hold promise as a treatment for more advanced Alzheimer’s, for which there are currently no therapies.—June Kinoshita
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World Alzheimer Conference 2000: Hot Topics Symposium
Several late-breaking, dramatic results were presented at this final symposium of the meeting. Michael Hutton and colleagues from Mayo Laboratory, Jacksonville, which only a few days earlier had first publicly presented data showing tangles and neurodegeneration in an FTDP-17 tau (P301L) transgenic mouse, revealed that double transgenics expressing the Swedish APP mutation and P301L tau also had NFTs and neuropathology, as well as Aβ plaques.
Yong Shen of Sun Health Research Institute described a successful method to culture neurons harvested postmortem from sporadic AD brain. He noted that the AD neurons developed fewer processes but, curiously, lived longer than neurons cultured from normal adult brains. It will be of great interest to examine the molecular and cell biology of the AD neuronal cultures, and they offer an intriguing possibility for drug assays.
David Morgan from University of South Florida presented data showing that Aβ vaccination blocked cognitive deficits in PS/APP double transgenic mice. Using a novel spatial learning task-a six-arm radial water maze-the USF investigators found that 12-month-old and 15-month-old mice that had received a course of vaccinations from four months of age onward performed as well as control animals, learning to navigate the maze with few errors. In contrast, age-matched nonimmunized mice were unable to learn the task and performed at chance. What’s more, 15-month-old mice that received a shorter course of vaccinations also improved to almost the same performance level as the controls. Fascinatingly, the 15-month-olds that had short-term vaccination had almost no reduction in Aβ burden compared to nonimmunized mice, suggesting that the plaques per se were not responsible for the observed cognitive deficits. One possible explanation is that immunization is leading to clearance of solube Aβ, which is causing the impaired function.
One hypothesis to emerge from the Aβ vaccination studies is that plaques may accumulate in Alzheimer’s patients because they are unable to clear Aβ as effectively as people who don’t get Alzheimer’s. Yangsheng Du presented data to support this notion, showing that CSF from Alzheimer's patients had a 30% reduction in levels of Aβ antibodies.
Miguel Pappolla presented data showing that high doses of melatonin produced dramatic drops in Aβ in the Tg2576 ("Hsiao") mouse. The mice were given around 1.5 mg per day of melatonin from age four months onward, and sacrificed at 15 months. The melatonin also produced "significant improvement" in performance on the Morris water maze, Pappolla reported.
In the final talk, Barry Reisberg presented preliminary results from the first U.S. clinical trial of memantine, a drug that targets the NMDA receptor system and which has been marketed in Germany. In the study, 126 patients with moderately severe to severe Alzheimer’s received 10 mg doses of memantine twice a day, while 126 matched controls received placebo. After six months, the treated patients were found to decline more slowly than the placebo group on a number of standard measures (which I was unable to note down because the data flashed by so quickly). Thus memantine may hold promise as a treatment for more advanced Alzheimer’s, for which there are currently no therapies.—June Kinoshita
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World Alzheimer Conference 2000: Amyloid Is Necessary and Sufficient to Cause Dementia
The scientific highlight of the Prof. Henry Wisniewski memorial symposium (12 July) was an informative lecture about familial British dementia (FBD) delivered by Blas Frangione. His studies with the 34 amino acid long ABri protein (abstracts 262 and 871) suggest that amyloid production alone is sufficient to initiate a cascade culminating in dementia. Antibodies against ABri recognize both parenchymal and vascular deposits and are accompanied with neurofibrillary changes very similar (both morphologically and biochemically) to those found in AD. Moreover, analysis of the Bri gene in a Danish kindred showed a 10 nucleotide duplication insertion which produces a frame shift and subsequent production of a 34 amino acid long amyloidogenic peptide (ADan). This second Bri mutation also causes dementia and presents with similar neuropathological changes. Thus, it appears that different amyloidogenic peptides can precipitate similar neurodegenerative processes and that amyloidogenesis is a key initiation event in these processes.—Dominic Walsh
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World Alzheimer Conference 2000: Aβ Vaccine Update
Last July, Dale Schenk and colleagues from Elan Pharmaceuticals reported the remarkable observation that simple immunization of PDAPP transgenic mice with amyloid-forming peptide sequences both prevents plaque formation and ameliorates existent plaques in brain (Abstract). PDAPP transgenic mice express a human amyloid precursor protein (APP) containing the FAD-associated V717F mutation under the control of the platelet-derived growth factor promoter. These mice show an age-dependent accumulation of extracellular amyloid plaques and an increase in astrocytosis. According to the amyloid hypothesis, amyloid buildup in brain is the primary cause of cognitive dysfunction in Alzheimer's disease (AD). Consequently, removal of such amyloid plaques should have beneficial effects in patients with AD. Schenk's results, therefore, suggested that it might be possible to remove amyloid plaques from human brain if similar immunization worked in humans.
At the meeting, Schenk reported on safety issues relating to the use of Aβ vaccination in animals, the mechanism by which vaccination removes plaques, and his group's progress towards developing a human Aβ vaccine. The safety of Aβ was studied in four different animal models: Swiss Webster mice, guinea pigs, rabbits, and monkeys. Schenk reported no adverse clinical manifestations related to Aβ immunization (including reactions at injection sites, overall clinical status, histopathology and effects on body weight). Moreover, in PDAPP transgenic mice, Aβ immunization reduced extracellular buildup of plaques but did not affect APP protein expression levels. Their results suggest Aβ immunization should not affect the normal physiological functions of APP, and that the immunization is selective for extracellular Aβ.
Schenk next showed that humoral-mediated (antibody-mediated) response, rather than T cell mediated response, is the most likely mechanism by which Aβ immunization removes plaques from brain. To prove this, they purified IgG antibodies from Aβ-injected animals and reinjected the purified antibodies into PDAPP transgenic animals at one-week intervals for six months. Amyloid burden was significantly reduced in the animals injected with IgG obtained from the mice injected with Aβ compared to animals injected with IgG purified from mice injected with an irrelevant antigen. Schenk subsequently showed that monoclonal antibodies specific for Aβ could also reduce plaque burden when injected into PDAPP transgenic animals.
The mechanism by which these anti-Aβ antibodies function appears to involve direct binding to antibody to amyloid within brain. The data suggest that a small amount of antibody must pass through the blood-brain barrier and enter the central nervous system. Schenk and colleagues used in vivo and cell culture systems to further show that the binding of antibody to amyloid deposits correlates with removal of those deposits by phagocytosis involving microglia. These results suggest that it may be possible to remove existent amyloid plaques in human brain using anti-Aβ antibody injections. The use of antibody injection therapy to remove amyloid deposits in human brain may be important since it is well-established that the immune response and antibody repertoire are both reduced during aging. Thus, for elderly AD patients who do not elicit an effective immune response, the use of antibody injections may circumvent potential problems relating to this inability of patients to elicit an Aβ immune response.
Finally, Schenk reported that his group had begun Phase I clinical trials, in both in the USA and the United Kingdom, to test Aβ immunization in patients suffering from mild cognitive impairment. So far, they report that vaccination of such patients with the Aβ formulation into humans is well tolerated and appears to be safe. The AD research community and world audiences now wait in anticipation for outcome of these Aβ immunization trials. It would be remarkable if immunization, which has been so effective in eradicating a number of human diseases such as smallpox and polio, may yet again prove to be a simple and effective way to prevent one of the most common and devastating human neurodegenerative disorders.
Further Note:
Several other groups have now replicated these Aβ immunization studies and have found that the procedure is effective in reducing amyloid plaques in different transgenic mouse models. At the special "Hot Topics" session held on July 13, David Morgan of the University of South Florida reported similar findings and, more remarkably, that Aβ vaccination also improved short-term learning behavior of APP transgenic mice despite little alteration of Aβ-plaque load in brain. In their studies, they used mice that were doubly transgenic for APP and PS1 (containing familial AD-linked mutations in both genes). These mice develop abundant Aβ plaques in an age-dependent fashion. Morgan and colleagues devised a modified Morris maze test, which typically involves mice learning to swim to one of several platforms on which food can be found. Instead, in their modified test they constructed six swimming alleys, akin to a maze, of which one ended at a hidden platform. The number of errors made by the mice in locating the hidden platform was used to determine if the mice had intact or defective memory. In essence their test measures short term spatial memory. After a learning period, normal non-transgenic mice are able to find the platform with less than one error in the fifth trial. In contrast, APP-PS1 transgenic mice at 15 months of age, which by this time have numerous plaques in brain, perform poorly in this test.
Morgan's group then studied the effects of Aβ immunization on this short-term spatial memory task. Transgenic APP mice immunized with Aβ for several months showed no deficits in learning, and were able to efficiently find the platform. In contrast, age-matched APP transgenic mice injected with control carrier keyhole limpet hemocyanin polypeptide, performed poorly in the test. Immunization with Aβ improved maze test performance in both APP and PS1 doubly transgenic mice as well as APP single transgenic mice. Surprisingly, even APP transgenic mice immunized with Aβ for a very short period also showed improved memory despite no noticeable change in Aβ accumulation within their brains. Their results suggest that, at least by this one memory test based on performance, Aβ immunization improves memory and that this can occur in the absence of any change in Aβ plaque deposition. Additional studies are needed to determine if indeed Aβ immunization improves memory assayed by other criteria, and whether Aβ plaque removal from brain is essential for improving memory.
Robert Malinow from Cold Spring Harbor Laboratories presented evidence (345) that β-amyloid is secreted during neuronal activity and may function in a negative feedback loop to block further synaptic transmission. Malinow showed that treatment of organotypic hippocampal slice cultures with agents that block neuronal activity, such as tetrodotoxin and high concentrations of magnesium, cause a 50%-70% reduction in secretion of Aβ into the medium. In contrast, agents that increased neuronal activity also increased Aβ secretion. He also demonstrated that wild-type and FAD mutant APP genes cause a 30% reduction in synaptic transmission when transduced into neurons. In contrast to these genes, both of which can be processed to release Aβ into the medium, transduction of a mutant APP gene, which cannot be processed into Aβ, has no effect on synaptic transmission. These results suggest that there is a complex interplay of APP processing and indirectly suggests that APP cleavage products have direct effects in modulating synaptic activity. The diverse roles of APP, including Aβ and its other cleavage products have been well-established, especially of their roles in growth control, calcium regulation, and apoptosis. The success of Aβ therapies will depend in large part on whether Aβ can be successfully eliminated from brain without compromising its other functions.—Mervyn J. Monteiro
Note: by Dominic Walsh
The aim of these studies is primarily to determine the safety of the vaccine in humans and to optimize the formulation so as to induce a good immune response. However, it is the outcome with respect to changes in mental status that will be awaited with great interest.
Although already in clinical trials there are a number of burning questions, which remain unanswered with regard to the usefulness of Aβ immunization. The PD-APP mouse is a good model for amyloid deposition; however, it is not a particularly good model for AD. While immunization is capable of removing amyloid plaques, what effect if any will it have on prefibrillar species? (For a discussion of the role of prefibrillar species in neurodegeneration, see the commentary on Peter Lansbury’s presentation.) It is possible (although I hope unlikely) that immunization by removing fibrils may effectively elevate the level of prefibrillar species and thus accentuate rather than alleviate Aβ-induced toxicity. Another, but less worrisome, possibility is that, if intracellular Aβ either directly or indirectly induces toxicity, then vaccination will not be able to alter that process. Over the coming months these important issues should be addressed in experimental models.—Dominic Walsh
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World Alzheimer Conference 2000: It’s Fibrillogenesis, Not the Fibrils!
Peter Lansbury, like I and others, has long promulgated the idea that intermediates in CNS disease-related fibrillogenesis processes are likely to be pathogenic, whereas mature matted fibrils (although not desirable) are relatively inert. In a provocative presentation Peter shared new data on the fibrillogenesis and pore forming properties of α-synuclein (α-S). Using AFM he demonstrated that, like Aβ, fibrillogenesis of α-S involves a transient population of oligomeric prefibrillar spheres (primitive protofibrils) and short flexible beaded fibrils (protofibrils, PF) and, under certain circumstances , annular PF.
Protofibrils, like fibrils, are β-sheet-rich structures, whereas monomeric α-S is natively unfolded (random coil). Incubation of monomeric α-S with certain artificial and brain-derived membrane vesicles revealed a weak association of the monomer with the membrane that was accompanied by a partial refolding to produce a helical-rich structure. In contrast, PF were found to bind avidly to membrane vesicles and to retain their strong β signature. Moreover, when PF and membrane vesicles loaded with the Ca2+-sensitive dye Fura 2 were incubated in the presence of extra-vesicular Ca2+, an increase in fluorescence was observed indicating that binding of PF resulted in an increase in membrane permeability to Ca2+.
Interestingly, incubation of primitive PF in the presence of brain-derived membranes produced annular PF which were detected on the vesicular surface by AFM. This observation leads Lansbury to speculate that annular PF act as pores and thus by "non-specifically" increasing membrane permeability lead to a decrease in neuronal viability. While not definitive, the data presented provide a plausible explanation as to how α-S PF may mediate toxicity in PD. Importantly, they explain why neurons with large fibrillar inclusions remain viable, whereas large numbers of neurons die without leaving significant amounts of fibrillar deposits.—Dominic Walsh
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World Alzheimer Conference 2000: Nearing Home BACE?
There were a number of oral and poster presentations relating to BACE biology (abstracts 313, 504, 540, 1013, 1269 and 1272). As with the original descriptions of BACE, the reports presented at this meeting were in broad agreement. Undoubtedly the most noteworthy presentation was by Martin Citron, but for convenience I have synthesized the findings of a number groups and listed them below.
- BACE is expressed on the cell surface, but undergoes N-terminal processing and glycosylation in the Golgi and accumulates in endosomes.
- Active site-directed mutagenesis (at Asp 93 or Asp289) did not alter pro-peptide cleavage of BACE indicating that this is not an endoproteolytic event.
- Pro-peptide cleavage appears to be mediated by Furin as active BACE is not produced in Furin-deficient cells, but can be induced by transfection of such cells with Furin.
- BACE is N-glycosylated at three of four possible sites.
- BACE contains three intramolecular disulfide bonds between the Cys pairs: 330-380, 278-443 and 216-420. Disruption of any of the three disulfides results in a loss of enzymatic activity indicating that all three bridges play a critical role in maintaining the required 3D conformation.—Dominic Walsh
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World Alzheimer Conference 2000: Presenilin's γ-Secretase Activity
Almost 12% (152) of the 1,307 abstracts presented at the 7th International Conference on Alzheimer’s Disease and related disorders made reference to presenilin (PS). For an overview of PS biology check out the webcast of Bruce Yankner’s plenary session lecture at: www.alzheimer2000.org/news/webcast.htm. This news report will limit itself to a summary of data presented which suggest a role for PS as γ-secretase (Abstracts: 311, 312, 313, 524, 603, 614, 635, 820, 821, 828, 837, 1267, 1270, 1274).
- Cells lacking both PS-1 and PS-2 do not secrete detectable levels of Aβ40 or Aβ42 and show a marked increase in APP C-terminal fragments (CTFs).
- Mutation of Asp257 or Asp385 of PS-1 prevents PS endoproteolysis, causes accumulation of APP CTFs and drastically reduces Aβ secretion. Mutation of homologous aspartate residues in PS-2 have a similar effect.
- APP CTFs (the direct substrates for γ-secretase) can be co-immunoprecipitated with both PS-1 and PS-2. These complexes accumulate when PS is inactivated (either pharmacologically or by mutation) and are readily detectable in the principal intracellular sites of Aβ generation.
- PS-1 co-elutes on size exclusion with a partially purified γ-secretase activity (obtained from solubilized membranes) as part of a protein complex.
- SDS-PAGE and subsequent silver staining of immunoaffinity purified γ-secretase activity (derived from solubilized membranes) identified hetrodimeric (i.e., NTF + CTF) PS and six other protein bands.
- "Naked PS-1" does not possess γ-secretase activity, indicating that it requires a number of critical cofactors for enzymatic activity.
- Potent γ-secretase inhibitors designed to function as transition state analogue inhibitors of aspartyl proteases (produced and characterized by two different groups) affinity label heterodimeric forms of PS-1 and PS-2.
- A biotinylated analogue of the general aspartyl protease inhibitor, Pepstatin A, also labels both PS-1 and PS-2.
- Small amounts of PS-1 are detected on the cell surface of neurons at tight junctions.
- PS-1 shares a similar sequence motif around Asp 385 as that seen in a family of bacterial proteases.
Although, as Colin Masters rightly said, "The pendulum has now swung in favor of the hypothesis that PS is γ-secretase," there still remain unanswered questions about the role of PS in amyloidogenesis. For instance, Robert Doms (Abstract 313) showed that cortical neurons from PS-1 KO mice display a dramatic decrease in secreted Aβ, but little or no change in intracellular (formic acid extracted) Aβ. Does this lack of change in intracellular Aβ in PS-1 KO mice mean that PS-2 is normally responsible for production of this pool of Aβ, or is there another γ-secretase activity responsible for the production of the insoluble intracellular pool of Aβ? This question will be readily addressed by comparing intracelluar pools of Aβ in PS-1 Vs PS-2 KOs and in PS-1 and PS-2 double KOs. Interestingly, Katja Fletcher (Abstract 1274) indicated that certain of her γ-secretase inhibitors could alter secreted and intracellular Aβ differentially. Given the recent recognition of the importance of intracellular Aβ any anti-secretase-based therapy should inhibit production of intracellular and secreted pools of Aβ. Note: Bristol-Myers Squibb is currently in phase I trials with a potent γ-secretase inhibitor which has no cross-reactivity with any other known protease and seems to be well-tolerated in humans. Importantly this γ-secretase inhibitor is more effective (by three orders of magnitude) at inhibiting cleavage of APP than inhibiting cleavage of Notch.—Dominic Walsh
(Editor’s note: Dr. Walsh is an author on Abstract 312, reported on in this news summary.)
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World Alzheimer Conference 2000: Pin1 Binds Both Tau and APP
One of the great unsolved mysteries of Alzheimer's disease is the link between the disease's two major lesions: Aβ amyloid plaques and tau tangles. Proponents of the amyloid cascade hypothesis presume that the former causes the latter. A fascinating alternative scenario is emerging from the laboratory of Peter Davies and his colleagues, which suggests that pathogenic changes to APP and tau may instead both be triggered by a single underlying event (Abstract 956). The Davies lab reported that phosphorylation of threonine 231 on tau results in a conformation that is strikingly similar to phosphorylation of threonine 668 on APP. Not only do many monoclonal antibodies raised against either of these two phosphoepitopes cross-react with both (and with no other phosphoepitopes), but both phosphorylated sites provide a substrate for Pin1, a molecule involved in regulation of mitosis. What's more, staining of Alzheimer's brain tissues with monoclonal antibodies shows that phosphothr 668 APP occurs in hippocampal and cortical neurons-and only in neurons that also stain for phosphothr 231 tau. The phosphothr 668 APP staining could clearly be seen in the lysosomal compartment.
What do these findings suggest? On the downstream side, Davies and colleagues previously have shown that Pin1 is present in Alzheimer's tangles, and that its binding to phosphothr 231 tau alters the conformation of tau, disrupting its ability to promote microtubule formation and possibly promoting PHF formation. Davies speculates that Pin1 binding to APP might result in increased Aβ formation. On the upstream side, Davies reported that cdc2 phosphorylates threonine 668 on APP, and suggested that a cdc2-like enzyme is involved in phosphroylation threonin 231 on tau. Others propose cdk-5 as a likely candidate. Much remains to be worked out, but one implication was clear: If Aβ accumulation and tau pathology are the result of parallel pathways initiated by a common trigger, then drugs targeting Aβ are not likely to halt tau pathology.—June Kinoshita
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World Alzheimer Conference 2000: Array Technologies in Alzheimer's Research
The relative paucity of presentations utilizing array technologies to study alterations of gene expression in Alzheimer's disease indicates that these powerful technologies are yet to be fully utilized in the study of AD. Three laboratories submitted abstracts using either cDNA or oligonucleotide arrays to examine message expression and one laboratory submitted an abstract using the recently developed protein chip array technology in combination with mass spectroscopy to examine a selected aspect of expression of Aß species..
Walter J Lukiw et al. from the LSU Neuroscience Center in New Orleans, LA, used Clontech cDNA arrays to compare the expression of 1184 genes in a pooled sample of message extracted from CA1 from five AD brains with a pooled sample of message from five control brains. The two samples were said to be matched for age (about 69), postmortem delay (about two hours), drug history, and agonal states. None of the AD brains sampled had a known history of familial neurodegenerative disease. Quantification utilized "control and alignment markers" that included α-tubulin, β-actin, HLAC1, HPRT, GAPDH, 60S ribosomal protein 13A, 40S ribosomal protein 9 and ubiquitin (many of which have in fact been shown to be up- or down-regulated in AD). Of the 22 largest differences detected, the AD sample showed significant decreases in the expression of genes encoding seven brain transcription factors and four messages involved in synaptic transmission, such as synaptophysin and ChAT. There were significant increases in message for six potentially proinflammatory genes, including β-amyloid precursor protein (bAPP), interleukin-1 (IL-1) precursors, and cytoplasmic phospholipase A2 (cPLA2). Lukiw suggested that these increases support the hypothesis of highly active neuroinflammatory processes operating in terminal AD hippocampal CA1.
In an additional presentation, Lukiw and Bazan utilized what appeared to be similar Clontech cDNA arrays of 1184 human cDNAs to evaluate recombinant IL-1ß induction of message expression by normal human neural progenitor (NHNP) cells in stable primary culture. They reported that IL-1β-induced cells showed 5.1-fold upregulation of message for cytoplasmic phospholipase and 9.1-fold induction of vascular endothelial growth factor. A 30-minute preincubation with the hetrazepine BN50730 suppressed these increases in cPLA2 and VEGF dramatically, and induced an independent set of genes including the gene encoding human anti-apoptotic factor Bcl-2.
Guilio M Pasinetti and his coworkers at Mount Sinai School of Medicine, New York, examined the expression of over 8,734 genes in brains of mild AD compared to cases with normal cognitive status. Twenty-five differentially regulated genes were identified and related to clusters of specific biological variations. Two such clusters were found to have patterns that correlated with variations in signal transduction pathways and cytoskeleton integrity. Pasinetti suggests that these early molecular markers of AD may correlate more closely with neuropsychological indicators of early AD than traditional neuropathologic measures of plaques and tangles.
Coleman et al. from the University of Rochester utilized home-made cDNA arrays, Clontech cDNA arrays and Affymetrix oligonucleotide arrays to examine message induction of homogenates and of single cells in AD and control brains. Ante-mortem blood samples were also examined. Brain regions examined were CA1 and superior frontal gyrus. Rather than utilize single messages as loading controls they compared the expression level of each gene in the arrays to every other gene in the arrays. They also analyzed the data using multivariate canonical analyses. The data showed: 1) that more genes are up-regulated than are down-regulated in AD. However, total message level is decreased in AD because some genes are down-regulated many-fold more than is the case for up-regulated genes; 2) a large number of genes related to the cell cycle were induced in AD, as were genes related to inflammatory responses. Down-regulated genes included ones related to neuronal plasticity (e.g., GAP-43), synaptic function (e.g., synaptophysin) and structural (e.g., actin) and other "housekeeping" activities (e.g., GAPDH); 3) multivariate analysis of expression data distinguished AD from control samples on the basis of homogenates of superior frontal gyrus, single cells from hippocampal CA1 and blood samples.
Message induction is one step in the progression from DNA to the delivery of a posttranslationally modified protein to its site of action. A single application of a relatively new protein chip technology was presented by Brian M. Austen et al. from St. George's Hospital Medical School, and Ciphergen, all of United Kingdom. This technology utilizes the binding of proteins to aluminum chips. The chips can be prepared to capture molecules on the basis of molecular weight, hydrophobicity, antibody binding or a variety of other properties. The bound species are then subjected to mass spect analysis to detect the m.w. of species captured on the chip. This method allows the discrimination of several hundred protein species, whose identity must be established by further analyses. This technology was used by Austen et al. to examine the effect of cholesterol on the production of Aß species by APP transfected HEK cells. In this study, antibody to the N-terminal 10 residues of Aβ was covalently linked on the ProteinChips. Mass analyses permitted detection of 12 discreet Aβ fragments, with 1-40 being the major species. The ProteinChip was also used to show that Aβ1-42 is the major variant in AD brain homogenates. Aβ1-40 secreted from cells was increased by preincubation with exogenous cholesterol (200mg/ml), and decreased by preincubation with lovastatin (50mg/ml).
In summary, array technologies are still being sparsely utilized. They are providing data that are consistent with data in the literature that have been obtained by other means. New molecules involved in the molecular pathology of AD are also being detected, but their identity is often either not yet determined or not revealed. There are also cases in which results from array technology are in conflict with data in the literature, a situation that emphasizes the need for confirmation of selected findings from arrays by other quantitative means, including quantitative in situ hybridization or real-time quantitative RT-PCR.—Paul Coleman
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World Alzheimer Conference 2000: Presentations on Mild Cognitive Impairment
Mild cognitive impairment (MCI) has been defined as a clinical entity whose characteristics were reviewed by Ron Petersen. Persons with MCI have memory impairment beyond what would be expected for age, yet they are not demented. These persons have relatively normal general cognitive function and activities of daily living. When compared to age and education-matched normal persons, measures of learning and delayed recall are significantly impaired. Their CDR will be 0.5.
Other investigators examined selected correlates of MCI. For example, Devanand et al. described olfactory identification deficits in patients with mild cognitive impairment (does this reflect a memory or an olfactory problem?) while Wolf et al. proposed that asymmetrical hippocampal atrophy may be a sign of MCI. Jack et al. reported that patients with MCI have hippocampal volumes which lie intermediate in size between controls and patients with probable AD. Blomberg et al. examined the ability of CSF tau, Aβ40 and Aβ42 to distinguish MCI from AD. Substantial overlap between AD and MCI was observed in all these markers, a finding reflective of the overlap in neuropathological markers presented below. Olichney et al. reported on a late positive event-related potential that peaked at 600 ms in response to a word repetition paradigm. In normals, the evoked potential was large in response to new words, and was greatly diminished with repetition. Fourteen patients with mild cognitive impairment had a smaller word repetition effect with a delayed onset of evoked response. These authors suggest the source of this potential was in the hippocampus. Mufson et al. continued with their studies of neurons with immunohistochemically defined chemical phenotype. Among their findings was the total number of trkA-ir neurons declined significantly from normal to MCI to AD (F92,27)=17.38;p.0.5). A global measure of cognitive function which combined all test scores was significantly related to the number of trkA-ir neurons (Spearman rank correlation=0.38, p=0.048). ANOVA revealed that the total number of p75NTR-ir neurons was significantly decreased in subjects with MCI (31 percent, pThere is no doubt as to the legitimacy of this relatively recently defined constellation of clinical signs. However, several questions arise as to the meaning of this clinical entity. These include: What is its prevalence? Does it represent an early form of clinically diagnosed AD that will inevitably progress to the full blown disease? What are its neuropathological, neurobiological and molecular correlates? Thirty-eight abstracts were devoted to these and other issues related to MCI.
Prevalence of MCI:
Relatively few presentations focused specifically on the prevalence of MCI. Hanninen et al., in a population study of 806 persons between the ages of 60 and 76 in Finland, reported that 52 subjects, 6.5 percent, met the MCI criteria. This percentage is in contrast to the 18-35 percent over age 65 cited by Bullock (referenced to Callaham, 1995). This difference may be a reflection of differences in inclusion criteria. In an extension of other studies Hallikainen et al. reported that women diagnosed as MCI had used estrogen replacement significantly less than cognitively better functioning women (5.5 percent versus 17.5 percent, p=0.004).
Progression of MCI to AD was a topic that attracted the greatest number of presentations. Issues covered dealt with the rate of progression in a sample and characteristics that might be predictive of conversion. Hanninen et al. reported that in a population sample of 806 subjects in Finland, more than 50 percent of cases classed as MCI converted to AD within three to four years. This percentage is in approximate agreement with Petersen's report of a conversion rate of 10-15 percent per year as compared to a rate of 1-2 percent per year for normal subjects of the same age. In further agreement, Nordberg et al. reported a conversion rate of 26 percent over two years. On the other hand, Bullock et al. reported a conversion of 32 percent over a one-year period in a cohort of 88 patients diagnosed with MCI (as defined in the European Consensus Guidelines). Of those that remained stable, the history in some suggested possible vascular aetiology and in others was not suggestive of AD, suggesting that the difference from the Petersen and the Hanninen data may be a consequence of differing definitions of MCI. The issue of differing inclusion criteria was also pointed out by Sadovnick et al. with some criteria having no stipulation of CDR staging.
Petersen suggested that certain features of the initial clinical presentation of MCI subjects, such as apolipoprotein E status, memory performance profile, and MRI-based measurements of the hippocampus, predict which individuals will progress more rapidly from MCI to AD.
Predictors of conversion:
Bennett et al. compared rates of change in global cognitive function (based on 20 cognitive tests) in 159 persons with mild cognitive impairment (MCI) to 464 persons with no cognitive impairment (NCI) and to 63 persons with early AD. The correlation between initial global score and rate of change was r=.17. However, for a memory score the correlation between initial level and rate of change was r=.54. In contrast, Sano et al. assessed seven symptom categories: memory, performance, disorientation, language, depression, behavior, and psychosis in a sample of 67 MCI patients who were followed at six-month intervals. Memory complaint, the most common symptom of individuals with MCI, was not predictive of conversion to AD. However, other specific symptoms, such as disorientation and the total number of symptoms were associated with subsequent conversion to AD.
The use of MRI data as predictors of conversion led to more agreement among studies than did use of psychometric tests. Jack et al. reported that measurement of hippocampal volume can provide predictive information as to which MCI patients will remain cognitively stable and which will convert to AD within a three- to five-year follow-up. Close correlation was seen between the rate of hippocampal atrophy and the probability of clinical decline. Murtha et al. reported a preliminary analysis indicating no difference in atrophy between Time 1 and Time 2 between elderly control subjects and those MCI subjects who did not convert to AD. The converters had a significantly greater change in atrophy than the control group and the nonconverters.
The data of Rossoret al., although not targeted specifically at MCI, are also consistent with a utility of MRI in predicting conversion. Using MRI to examine a cohort of at risk persons with a history of familial Alzheimer's disease with APP and presenilin 1 mutations revealed a presymptomatic phase of tissue loss in patients who subsequently develop the disease.
PET data, especially glucose measures, proved to be uniformly useful in predicting conversion. Nordberg et al. reported that deficits in glucose metabolism predicted clinical outcome in 93 percent of their cases. Mony De Leon reported data from a four-year longitudinal study assessing regional cerebral glucose metabolic (CMRglu) and neuropsychologal predictors of MCI conversion to AD. Subjects received a baseline diagnostic examination that included FDG-PET, MRI and memory tests. FDG-PET scans were coregistered with MRIs and CMRglu was obtained from four subregions of the temporal lobe, the supramarginal gyrus, two subregions of the frontal lobe, the posterior cingulate gyrus and the pons. Compared to the normal control group, the MCI group that converted to AD showed, after scan normalization, widespread metabolic reductions in the hippocampal formation and the temporo-parietal area (>20 percent). Significant reductions in immediate and delayed memory performance were also noted. There were no significant CMRglu or cognitive differences between the MCI group that remained stable at follow-up and the normal control group. Both CMRglu and memory performance predicted conversion to AD. De Leon also reported that entorhinal cortex changes in normal elderly predict the hippocampal and memory changes characteristic of MCI. Hippocampal changes in MCI predict neocortical changes and the clinical diagnosis of AD. Thus, these data indicate that over four years, conversion from MCI (as defined in the de Leon studies) to AD is not inevitable, and that it is possible to predict who may remain stable.
There were also isolated reports of other measures with potential for predicting conversion from MCI to AD. Olichney et al. reported on a positive event-related potential that peaked at 600 ms in response to a word repetition paradigm. Analyses, comparing MCI cases to normals showed that whereas normals and nonconverters showed a reduction of evoked response with repetition, converters did not show this reduction. These authors suggest that, albeit based on a small sample, differences between MCI convertors and nonconvertors indicate that this evoked response measure may be particularly sensitive to early AD pathology. In a separate study Blennow et al. found increased CSF-tau and decreased CSF-Aβ42 levels in MCI patients, to be predictive of conversion to AD with a sensitivity of 88 percent, and a specificity of 80 percent. These data are in stark contrast to the data of Blomberg et al. reported above in which substantial overlap between AD and MCI was observed in these CSF markers.
Although not directly related to MCI, Coleman et al. reported that levels of synaptophysin message in single postmortem neurons declined progressively with immunohistochemical markers of disease state. These data suggest that a longitudinal PET study of a synaptic marker may reveal a quantitative indicator of probability of conversion from MCI to AD.
Neuropathology of MCI:
The neuropathological status of persons with MCI extended over a wide range of involvement when using traditional indices of plaques and neurofibrillary tangles. Joseph Parisi presented neuropathological data from nine brains of persons who had been classified as MCI at the Mayo Clinic, Rochester, Minnesota. Although these nine patients had a single diagnosis of MCI, they presented different neuropathological pictures. Several fell in the range of Braak III-V with plaques that were mostly diffused with a few neuritic plaques. Several more of these MCI cases were Braak II-III with no plaques and were considered to have argyrophilic grain disease. Finally, one case had neurofibrillary tangles only in the medial temporal lobe and was classified as having had NFT-only disease. The finding of a range of Braak scores in a set of cases with a similar level of cognitive impairment is consistent with the consensus report on neuropathological diagnosis of AD that gave a probabalistic definition of the relation between Braak stage and dementia. Bennett reported postmortem data on 85 persons: 27 with MCI, 37 with no cognitive impairment, and 21 with AD. A global measure of AD pathology was created using the average z-scores for neuritic plaques (NP), diffuse plaques (DP), and neurofibrillary tangles (NFT) from three neocortical regions. AD pathology increased linearly across the three groups but accounted for only 23 percent of the variance. This conclusion is consistent with the Mayo Clinic data reported by Parisi (above). Bennett suggested that both the course of cognitive decline and severity of AD pathology in persons with MCI is intermediate to that of NCI and AD, and that performance on tests of episodic memory may be a better predictor of future decline than global cognitive function.
In summary, MCI represents a clinical definition with variable underlying pathology and a primary hippocampal focus. The rate and the probability of conversion to AD, although statistically predictable in a large enough sample, shows considerable individual variability that is probably reflective of variability in underlying pathology and, perhaps, differing basic neurobiological mechanisms. Thus, the present clinical definition of MCI may represent a variety of underlying mechanisms all leading to the final common path of MCI.—Paul Coleman
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World Alzheimer Conference 2000: Evolving Brain Abnormalities in AD
Reported by Paul Coleman
Bradley T. Hyman and Paul D. Coleman, Chairs
This symposium (held 10 July) covered the gamut of age-related evolution of brain function in AD from behavioral studies emphasizing mild cognitive impairment (MCI) presented by Steve Ferris through microscopic structure (John Morrison, Bradley Hyman and Mark West) to events at the cell biological and molecular levels (Eva-Maria Mandelkow and Paul Coleman).
Steve Ferris outlined the battery of psychometric tests being used at the New York University Alzheimer's Disease Center. He presented data indicating that the NYU Paragraph Recall Test was useful in identifying cases at high risk of being diagnosed with AD within several years. In their experience, the rate of conversion from MCI to diagnosed AD was 10%-15% per year-a figure that corresponds exactly to comparable data from Petersen at Mayo Clinic, Minnesota.
This description of behavioral data was followed by three presentations centering on structural aspects of dementia that were not closely related to MCI, but that did deal with structural changes that differentiated AD from normal aging. Mark West outlined the procedures of unbiased stereology for counting total numbers (not density) of neurons and showed data demonstrating that, whereas in AD there was a major loss of neurons in hippocampal CA1, there was no such loss in normal aging. These data led West to conclude that AD is qualitatively different from normal aging and, therefore, not an extension of healthy aging. John Morrison stressed the chemical phenotype of neuron classes that were among the earliest to decline in AD. He reported that neurons expressing the NR1 receptor were exquisitely sensitive indicators of AD pathology. Bradley Hyman demonstrated neuron loss in thioflavin positive plaques, but not in plaques that were not thioflavin positive (see News Report).
The symposium then shifted to a more molecular focus. Eva-Maria Mandelkow presented data from their continuing investigations of the cell biology of tau. She showed data demonstrating that the kinase, MARK, phosphorylates Ser 262 in the microtubule binding domain of tau, as well as KXGS motifs in MAP2 and MAP4. The hyperphosphorylation produced by overexpression of MARK in vitro leads to massive disruption of microtubules and cell death. On the other hand, MARK phosphorylation of KXGS motifs in tau are essential for tau-induced process formation in model cell systems, pointing to the requirement for a delicate balance in MARK activity. Additional studies of the cell biology of tau showed that tau is capable of inhibiting kinesin-dependent transport in the plus direction of microtubules. Tau does not change the speed of transport, but does decrease the probability of attachment of motors to microtubules. In the case of nerve cells mitochondria and other organelles then disappear from the cell processes, which suggested to Mandelkow consequent energy deprivation and vulnerability of cells.
This symposium concluded with a presentation by Paul Coleman of work from his laboratory dealing with message expression of single cells and homogenates in Alzheimer's disease. Quantitative in situ hybridization of synaptophysin message in conjunction with double immunohistochemistry for neurofibrillary tangles and tau phosphorylated at selected epitopes demonstrated an incremental loss of synaptophysin message progressing from immunonegative neurons in control brain to immunonegative neurons in AD brain to tangle-free neurons immunopositive for phospho tau at Ser 262 to neurons with frank neurofibrillary tangles. Phosphorylation at Ser 396/404 resulted in no additional decrement of synaptophysin message in NFT-free neurons. The decreased expression of synaptophysin message was not a consequence of a generalized decrease in message expression since message for cathepsin D was increased in tangle-bearing neurons-in agreement with data of Nixon and Cataldo.
These, and other, demonstrations that although some message levels decrease in AD other message levels increase leads to the concept of changing profiles of gene expression as disease progresses, rather than a generalized decrease of message expression. The advent of array methods to profile expression of message of large numbers of genes, either in homogenates or in single, immunohistochemically defined cells, provides the opportunity to examine these profiles of message expression in greater detail than previously possible. Coleman presented data from some of the array studies from his lab. The general conclusions from these data are: 1) more genes show increased and fewer genes show decreased expression in AD; 2) gene classes showing increased expression in AD include genes related to the cell cycle and genes related to inflammatory processes. Gene classes showing decreased expression in AD include those related to selected aspects of synaptic and cell structure and function and those related to metabolic activities. Note that these latter two classes include genes such as actin and GAPDH which are often used as "control" genes; 3) multivariate statistical analyses separate AD samples from control samples on the basis of expression of appropriate sets of genes.
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World Alzheimer Conference 2000: Synuclein Update
Reported by Benjamin Wolozin, Loyola University Medical Center www.meddean.luc.edu/lumen/DeptWebs/pharm/wolozin.htm
Increasing attention has focused on the role of synucleins in neurodegeneration. α-synuclein was originally cloned by Richard Scheller from rat brain (1). Subsequently, it was cloned by Julia George and David Clayton from the finch brain (2), and by Ueda, Masliah, Saitoh and colleagues from human brain (3). At the oral session titled, "The Synucleins and Lewy Body-Related Diseases," Julia George reviewed some of the fundamental biology of α-synuclein (1291) (4). She pointed out that it exists as a natively unfolded protein, but develops helical structure upon exposure to acidic phospholipids (5). In most cases, α-synuclein exists as a monomeric protein in the α-synuclein/lipid complexes. However, certain phospholipids are able to induce oligomerization of α-synuclein into SDS-stable complexes containing two to four α-synuclein molecules (1291). This raises the possibility that the interaction of α-synuclein with particular cellular lipids could influence its pathological aggregation. Earlier in the meeting, Sharon, Goldberg and Selkoe presented evidence indicating that some membrane-bound α-synuclein (from brains of transgenic mice and MES23.5 cells) can be detected as higher molecular weight oligomers, although most of the α-synuclein exists as a cytoplasmic, monomeric protein (52). John Trojanowski's presentation reviewed research on the pathology of the synucleins (1290, also see www.med.upenn.edu/cndr) (6). α-synuclein has been shown to accumulate in a variety of forms in multiple different diseases. α-synuclein accumulates in Lewy bodies in a variety of diseases such Parkinson's disease, diffuse Lewy body disease and familial Alzheimer's disease. Lippa and colleagues showed evidence suggesting that the presence of Lewy bodies in familial Alzheimer's disease is associated with unusual clinical presentations that include visual hallucinations, fluctuating mentation and other psychotic features (173). Trojanowski also showed that α-synuclein is associated with other types of pathologies (1290). Neurites in multiples diseases including Parkinson's disease, diffuse Lewy body disease, familial Alzheimer's disease and neurodegeneration with brain iron accumulation type I (1290, 240). Glia can also develop accumulation of α-synuclein in multiple systems atrophy and in amyotrophic lateral sclerosis. Thus, α-synuclein aggregates are not restricted to neurons. Trojanowski's group has also shown that β- and c-synuclein also accumulate along with α-synuclein, but the amount of β- and c-synuclein that aggregate are much less than that for α-synuclein. The amount of aggregation appears to correlate with the inherent tendency of the proteins to aggregate, because studies show that β- and c-synuclein do not aggregate as readily as α-synuclein in vitro (1290).
The mechanisms underlying synuclein aggregation in neurodegeneration remain controversial, but it is becoming increasingly clear that protein oxidation plays an important role. Harry Ischiropoulos presented his research investigating the putative role of tyrosine nitration in α-synuclein aggregation (1292) (7). α-synuclein has four tyrosines that could be substrates for nitration induced cross-linking. Ischiropoulos showed that α-synuclein could be nitrated and that peroxynitrate could induce cross-linking of α-synuclein in vitro. Although all four tyrosines in α-synuclein can be nitrated, Ischiropoulos showed that nitration of the three tyrosines at the C-terminus of α-synuclein is required for oligomerization of α-synuclein (1292). Peroxynitrate can also induce oligomerization of α-synuclein in cell culture; however, only a small number (~25%) of cells actually show α-synuclein aggregates under these conditions. Other studies from the group of Ischiropoulos, Lee and Trojanowski show that Lewy bodies contain 3-nitrotyrosine and nitrated α-synuclein, which indicates that α-synuclein is nitrated in brains of patients with Parkinson's disease (240). Whether nitration of α-synuclein is a primary or secondary event in its aggregation in neurodegeneration remains to be determined.
A different model for α-synuclein aggregation was presented by myself, Benjamin Wolozin (1293) (8). Previous studies by Hashimoto, Masliah and colleagues and by Paik, Kim and colleagues indicated that metals could induce oligomerization of α-synuclein in vitro (9, 10). Work in my laboratory shows that FeCl2 might also be an important inducer of α-synuclein aggregation. Neuroblastoma cells overexpressing A53T α-synuclein developed aggregates upon exposure to 0.3-10 mM FeCl2. Wild-type and A30P α-synuclein also developed aggregates but only when neuroblastoma cells or primary cortical neurons were treated with both iron and an oxidative generator, such as dopamine or hydrogen peroxide. The aggregates could be detected by immunoblot, immunohistochemistry, electron microscopy or thioflavine-S histochemistry. The amount of aggregate was dose dependent, being present in 100% of the cells with high-dose treatment and 25% of the cells at the milder treatments. As with the nitro-tyrosine story, the role of iron in α-synucleinopathies appears to have strong clinical relevance. Most, if not all, of the synucleinopathies appear to be associated with iron accumulation, and George Perry presented evidence indicative that reactive iron (FeII) can be detected in Lewy bodies (969) (11).
One of the most pressing needs in the field of synuclein research is identification and distribution of good animal models mimicking Lewy body diseases. Lee presented recent results obtained with a strain of transgenic mice overexpressing α-synuclein using a prion promoter (1294). Mice expressing A53T α-synuclein (but not wild-type or A30P α-synuclein) develop severe motor pathology at about 11 months of age. The behavioral characteristics of the mice have not been fully characterized, but comments from neurologists in the audience suggest that the behavior appeared to be dystonic. Lee had performed some preliminary analyses of pathology and observed the presence of α-synuclein accumulations in the deep nuclei of the cerebellum and in the brain stem, but no obvious pathology in the substantia nigra.
The transgenic α-synuclein mouse model that best resembles Lewy body diseases is that developed by Masliah, Mucke and colleagues (12). This mouse develops motor deficits at 11 months of age, shows loss of dopaminergic markers and the presence of α-synuclein accumulations in the substiantia nigra. Having developed this model, the group has begun to explore factors that modulate α-synuclein pathology. Masliah presented a new study in which they crossed the transgenic mice overexpressing α-synuclein with trangenic mice overexpressing β-synuclein (1295). They observed that overexpressing β-synuclein can reduce α-synuclein aggregation, pathology and motor deficits-the performance of the α/β-synuclein overexpressors was equal to that of control mice, although slightly below that of mice overexpressing β-synuclein alone. The aggregation-inhibiting ability of β-synuclein might result from a direct interaction between α- and β-synuclein, because Masliah showed that β-synuclein could also inhibit aggregation of α-synuclein in vitro. Although they have yet to show coassociation of α- and β-synuclein, it appears likely that there is some interaction between the two types of proteins.
In summary, α-synuclein has a strong tendency to aggregate. A consensus is emerging indicating that oxidative factors, such as tyrosine nitration or iron-induced oxidation, increases the tendency of α-synuclein to aggregate. The aggregation can be modified by interaction with other factors, such as homologous proteins (β-synuclein), which inhibits aggregation, or long chained lipids, which induce oligomerization. Aggregation of α-synuclein is likely to be dose-dependent because mice that overexpress α-synuclein develop accumulations of α-synuclein aggregates and associated motor deficits. A dose dependence of aggregation is also seen in vitro and in cell lines. Simply overexpressing α-synuclein, though, does not appear to be sufficient to mimic Lewy body disease because the resulting pathology that occurs in transgenic mice does not often mimic that seen in human neurodegenerative disease. Understanding the factors that increase α-synuclein aggregation, and the mechanism of toxicity of aggregated α-synuclein have emerged as pressing questions.
References:
52. Sharon R, Goldberg M, Selkoe DJ. Human α-synuclein exists in both monomeric and polymeric membrane-bound forms in transgenic mice and Mes23.5 dopaminergic cells overexpressing WT and A53T mutant α-synuclein.
173. Lippa CF, Nee L, Pollen D, Lee VMY, Trojanowski JQ. Lewy bodies may influence symptomatology in some cases of presenilin-related Alzheimer's disease.
240. Duda JE, Giasson BI, Chen Q, Souza JM, Murray IVJ, Ischiropoulos Lee VMY, Trojanowki JQ. α-synuclein nitration in neuropathological inclusion.
969. Perry G et al. Neurodegenerative disease: role of free radical damage.
1290. Trojanowski JQ. Overview of the synucleinopathies.
1291. George J. Synuclein and synelfin: the songbird story.
1292. Ischiropoulos H. Mechanisms of oxidative injury in neurodegenerative synucleinopathies.
1293. Wolozin B. α-synuclein in Lewy body diseases.
1294. Lee M et al. Synucleins: in vitro and in vivo studies.
1295. Masliah E. Mechanisms of synuclein and NAC fibrilogenesis.
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- Maroteaux L, Campanelli JT, Scheller RH. Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci. 1988 Aug;8(8):2804-15. PubMed.
- George JM, Jin H, Woods WS, Clayton DF. Characterization of a novel protein regulated during the critical period for song learning in the zebra finch. Neuron. 1995 Aug;15(2):361-72. PubMed.
- Uéda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11282-6. PubMed.
- Clayton DF, George JM. The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends Neurosci. 1998 Jun;21(6):249-54. PubMed.
- Davidson WS, Jonas A, Clayton DF, George JM. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem. 1998 Apr 17;273(16):9443-9. PubMed.
- Duda JE, Lee VM, Trojanowski JQ. Neuropathology of synuclein aggregates. J Neurosci Res. 2000 Jul 15;61(2):121-7. PubMed.
- Souza JM, Giasson BI, Chen Q, Lee VM, Ischiropoulos H. Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies. J Biol Chem. 2000 Jun 16;275(24):18344-9. PubMed.
- Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B. The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J Neurosci. 2000 Aug 15;20(16):6048-54. PubMed.
- Hashimoto M, Hsu LJ, Xia Y, Takeda A, Sisk A, Sundsmo M, Masliah E. Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro. Neuroreport. 1999 Mar 17;10(4):717-21. PubMed.
- Paik SR, Shin HJ, Lee JH, Chang CS, Kim J. Copper(II)-induced self-oligomerization of alpha-synuclein. Biochem J. 1999 Jun 15;340 ( Pt 3):821-8. PubMed.
- Castellani RJ, Siedlak SL, Perry G, Smith MA. Sequestration of iron by Lewy bodies in Parkinson's disease. Acta Neuropathol. 2000 Aug;100(2):111-4. PubMed.
- Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science. 2000 Feb 18;287(5456):1265-9. PubMed.
World Alzheimer Conference 2000: Presenilin Roundup
Reported by Mervyn Monteiro
2000 August 2. Presenilins and Lewy body pathology. Lippa et al (173) used α-synuclein immunoreactivity to determine the frequency of Lewy body (LB) occurrence in familial Alzheimer's disease (FAD) cases linked to mutations in PS1. Interestingly, they found that approximately half of 14 afflicted brains contained LBs. Moreover, some of the LB-contain brains were from deceased patients whose clinical records were consistent with their having had Parkinsonism-like symptoms. In an earlier report, this group found LBs in approximately 60 percent of brains from patients with FAD-linked mutations in PS and APP genes. Fishel et al. (294) also reported that they had found LB by α-synuclein staining in six of 11 brains from individuals having FAD-linked mutations in PS2. Taken together, these results suggest that a significant fraction of FAD-linked mutations in PS1 and PS2 lead to LB pathology. Insight into what causes these inclusions may provide insight into the etiology of the disease.
Presenilins and the cell cycle
Mice disrupted in the murine presenilin 1 (PS1) gene die on day one after birth. However, these mutant mice survive to adulthood when the human PS1 transgene is expressed under the control of the Thy-1 promoter. Zheng et al. (599) reported, however, that these rescued mice develop extensive epidermal hyperplasia and neoplasia. The abnormalities appear to arise due to the restricted expression of the Thy-1 driven PS1 transgene to neurons and its failure, unlike the normal murine PS1 gene, to be expressed in skin. Zheng et al. provided evidence for the lack of expression of the human PS1 transgene in keratinocytes cultured from the rescued mice. In these cultures, β-catenin protein had a longer half-life and cyclin D1 expression was increased, compared to keratinocytes expressing endogenous murine PS1. Edward Koo (16) also provided supporting evidence that loss of PS1 expression leads to increased cyclin D1 expression. Since increased β-catenin and cyclin D1 are both associated with induction of cellular proliferation (both proteins are involved in the G1 to S phase transition of the cell cycle), these data suggest that presenilins may have important roles in the regulation of the cell cycle and in growth control.
The unfolded protein response
The unfolded protein response (UPR) is up-regulated during cellular stress and when misfolded proteins accumulate in the endoplasmic reticulum (ER). A number of presentations, including those by Katayama et al. (1176), De Strooper et al. (342), and several others at the meeting, suggested that the UPR is compromised in cells disrupted in the PS1 gene, and in cells expressing mutant FAD PS1 genes. Furthermore, Katayama and colleagues reported that the chaperone protein BiP was decreased in AD brains (Nature Cell Biol;1(8):479-85). De Strooper also reported at the meeting that they too had observed down-regulation of chaperone proteins, BiP and GRP94, in cell cultures prepared from PS1 and PS2 knockout mice.
Contrary to the these findings, Sato and colleagues (1186) reported that the UPR response was unaltered in PS1-deficient cells and in cells expressing FAD-linked PS1 mutations. In fact, Sato et al. reported that PS1-deficient cells, as well as neuroblastoma cell lines stably expressing several different FAD PS1 mutations, are able to efficiently induce transcriptional up-regulation of genes involved in the UPR. Thinakaran induced cellular stress with three different agents: tunicamycin, thapsigargin, and a Ca2+ ionophore. Each of these treatments caused induction of BiP (GRP78), GRP94, and CHOP mRNA to equivalent levels and with a similar profile in wild-type PS1 expressing cells, in PS1-/- cells, and in cells expressing FAD mutant PS1 proteins. Moreover, Thinakaran et al. did not find any noticeable difference in BiP protein levels in protein lysates of transgenic mice carrying FAD PS1 mutant genes or in human brains from FAD individuals.
The calcium connection
At least three different calcium-binding proteins are known to interact with presenilins: calsenilin, calmyrin and sorcin. Wasco (19) described studies of calsenilin, which has highest homology to the recoverin family of proteins. Calsenilin has two structural domains: a long N-terminal domain and a C-terminal domain containing four calcium-binding EF hand motifs and. Calsenilin binds to both full-length presenilin and C-terminal fragments of presenilins. Calsenilin protein is soluble when overexpressed in cells. However, when it is coexpressed with PS2, calsenilin fractionates predominantly with the membrane-bound presenilins. Membrane binding of calsenilin was not affected by changes in calcium levels, as treatment with EGTA did not cause any noticeable release of calsenilin from the membrane-bound pool. Calsenilin is cleaved during apoptosis. This cleavage is sensitive to zVAD treatment, indicating it is likely due to a caspase(s) action during apoptosis. Purified caspase 3 was found to cleave calsenilin at a site between the N-terminal and EF hand. The N-terminal domain of calsenilin was found to mediate binding to presenilin.
Lilliehook et al. (1172) reported on the physiology associated with overexpression of calsenilin in cells. In one approach, they used microarray analysis to examine changes in mRNAs profiles expressed in calsenilin-induced and non-induced cells. The identity of a number of mRNAs that were down regulated upon calsenilin expression was described. Two of these were phosphoinoside-3-kinase class 3 and inositol 1,4,5-trphosphate receptor (IP3R), type I. In immunoblot experiments they confirmed that IP3R was reduced in cells overexpressing calsenilin. They next examined how calsenilin overexpression affects calcium responses in H4 neuroglioma cells. Calsenilin expression led to reduce amplitude of calcium release induced by carbachol (agonist) treatment compared with non-expressing cells. Calsenilin expression also caused an elevation in amplitude of calcium release induced by thapsigargin treatment, suggesting that calsenilin increases the calcium content of the ER. Moreover, calsenilin overexpression enhanced apoptosis induced by both serum withdrawal and thapsigargin treatment. Taken together, the data suggest that calsenilin decreases IP3-mediated calcium signaling and increases calcium content of ER stores.
A common feature associated with FAD mutations in both presenilin and APP genes is the dysregulation of calcium signaling.
Consistent with this observation, Kim et al. (534 and 633) presented evidence that presenilins carrying FAD mutations have altered capacitance calcium entry (CCE). Similar CCE defects were recently reported by Leissring et al. (JCB 149:793-797). CCE is the influx of extracellular calcium as a compensatory mechanism to replenish calcium depleted from intracellular stores. Kim demonstrated that cultured neurons from PS1 knockout embryos had elevated CCE responses compared to neurons cultured from embryos expressing wild-type PS1. Similarly, cells stably expressing the PS1 D257A engineered mutation, which acts as a dominant negative mutation in terms of Aβ secretion, had elevated CCE responses. Kim also presented data demonstrating that FAD mutations in PS1 and PS2 genes cause reduced CCE.
To determine whether there is a molecular link between CCE and Aβ production, Kim et al. treated cells with SKF96365, a CCE inhibitor. As expected SKF96365 inhibited CCE responses but interestingly this treatment correlated with an increase in Aβ production. Moreover, Kim presented findings that overexpression of the putative Drosophila CCE channel, Trp6, causes a reduction in Aβ production. Finally, Kim showed that addition of Aβ to cultures does not affect CCE. Taken together these results suggest that FAD presenilin mutations cause reduced CCE responses in cells and that this defect is likely to be upstream of the effect on Aβ production. Since FAD PS mutations have increased agonist-induced IP3 regulated calcium release, the combined effects of these two alterations may result in low cellular stores of calcium which may be detrimental to cell survival.
Presenilins and APP cleavage
Yankner et al., (603) reported that cells cultured from mouse embryos disrupted in both PS1 and PS2 genes do not undergo any detectable proteolytic cleavage of APP that corresponds to c-secretase activity. Thus, these cells fail to produce any detectable Aβ fragments. Instead, cells lacking both presenilins produce an increase in two other C-terminal APP cleaved fragments, C83 and C99 fragments, which are derived from α- and β-secretase cleavage, respectively (see Nature Cell Biol 2:463-5). Similar results were reported at the meeting by De Strooper et al. (see Nature Cell Biol. 2:461-2). These results are consistent with the notion that absence of presenilins eliminates c-secretase cleavage of APP. However, Yankner reported that there was a noticeable change in the proportion of the two C-terminal fragments of cultures prepared from mouse embryos disrupted in one versus both presenilin genes. In single PS1 knockout mice the proportion of the C99 species represented approximately 15%-30% of the total C-terminal fragments. In contrast in PS1/PS2 double knockout mice the C99 fragment was reduced to only 5% of the total fraction. These results suggest that loss of presenilin expression not only affects c-secretase cleavage of APP but also β-secretase cleavage of APP.
Steiner, et al., (303) reported on mutation analysis of residue 384 in PS1. Residue 384 of wild-type PS1 is a glycine. However, a FAD PS1 missense mutation causing a glycine to alanine substitution has been identified. Haass showed that cells expressing the presenilin FAD G384A mutation produce the largest documented increase in Aβ42 production. In contrast, mutation of this glycine to a number of other residues produced relatively small changes in Aβ production. They reasoned that since residue 384 was important for c-secretase activity, and it is adjacent to the aspartate 385, which is one of two aspartates (the other being D257) proposed to be essential for presenilin-associated c-secretase activity, it could be part of a signature sequence conserved in other proteases. They, therefore, compared the sequence surrounding residue 384 to see whether there was homology to any other protein in databases. Not only did they find the surrounding sequence to be highly conserved in all presenilin proteins across species, but they also found homology to a sequence present in proteins belonging to the type 4-prepilin-peptidase family. The type 4-prepilin peptidases (TFPP) are membrane-bound proteases present in different bacterial species, and like presenilins, are thought to constitute a novel family of bi-lobed aspartate proteases (see JBC 275:1502-10). TFPP cleaves the bacterial prepilin leader sequence releasing pilin protein into the periplasm. The processed pilin is then assembled into pili, which has diverse functions including, toxin secretion, gene transfer, adhesion and infection. Haass indicated that presenilins and prepilin both possess a conserved motif (G/AXGDX where X is variable and GD is the sequence at residues 384 and 385, respectively, in PS1). The similarity in the protease cleavage functions of presenilins and TFPP and the functional relationship will surely be explored in future studies.
Iwatsubo et al (631) described mutagenesis experiments of the sequence, ALPALP, which is a highly conserved in the C-terminal region of all known presenilins. The C-terminal proline in the sequence is mutated in FAD. They hypothesized that the conserved sequence could be important in presenilin function and, therefore, proceeded to mutagenize both proline residues. They reported that when the N-terminal most proline is mutated to a leucine in PS2 it does not affect either Notch cleavage or Aβ production. In contrast, mutation of the C-terminal proline to a serine abolishes both Notch and cleavage as well as Aβ production. They propose that the C-terminal proline of the ALPALP sequence may be important in the folding and stability of presenilins.
Presenilin interactors
De Strooper et al. (602) used a yeast 2-hybrid interaction trap to identify telencephalin as a interactor of the C-terminal region of PS2. Telenecephalin is a member of the NCAM superfamily of molecules, and is expressed in hippocampus and dentate gyrus. Telencephalin is also known to interact with integrins and has been functionally implicated in promoting neurite outgrowth. It has also been implicated in LTP. Interestingly, in presenilin knockout mice, telencephalin was abnormally localized, suggesting that lack of presenilins affects either sorting or localization of telencephalin.
Livne-Bar described a genetic screen for PS-interacting genes in Drosophila (928). They used a P-element screen to identify modifiers of PSEN defects, including those able to rescue a PSEN lethal phenotype. About two dozen interacting genes were isolated by this screen. These genes fell into three broad classes: genes involved in the Notch signaling pathway, genes involved in programmed cell death, and cell cycle-regulated genes. One of the genes able to rescue PSEN null larvae was shibire. Shibire whose mammalian counterpart is dynamin, belongs to the GTPase superfamily. Dynamin and shibire have been shown to play crucial roles in receptor-mediated endocytosis, although there is some contention as to whether dynamin functions as a mechanochemical motor or as a regulatory molecule required to activate downstream effectors involved in coated vesicle formation.
Ken Kosik (634) provided evidence that disruption of the Drosophila presenilin gene produces neurogenic defects and accumulation of large ubiquitin-positive inclusions within cells. In these mutant Drosophila embryos, Armadillo (β-catenin) is also mislocalized. Kosik et al. used microarry analysis to determine changes in RNA expression in the brains of mice lacking or containing presenilin genes. Out of over one thousand transcripts analyzed, only a handful were upregulated. Substantially more genes were downregulated, some of which included frizzled receptor, CaM kinase, SOX13, a Rad23 homologue, Nibrin, and interleukin 7.
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