Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part I

11 Apr 2003

Almost exactly 100 years after Alois Alzheimer saw his first patient who complained about "having lost herself," Christian Haass and Roger Nitsch invited a panel of international opinion leaders to gather in the German Black Forest for the 87th International Titisee Conference of the Boehringer Ingelheim Fonds and discuss current findings on molecular mechanisms, animal models, and, in particular, therapy of Alzheimer’s disease (AD) and Parkinson’s disease (PD). The romantic beauty of the surrounding scenery conferred a peaceful, yet spirited environment for the exchange of thoughts and ideas. Hot topics at center stage of discussion were sprinkled with exciting off-mainstream presentations of outstanding quality. Buoyed by blue skies, a general sense of optimism and a feeling that therapy for AD may come within reach spread throughout the meeting. In terrifying contrast with this peaceful paradise of mind, late-night images on CNN confronted the participants with the unfolding madness in the rest of the world. What follows are rapid notes made during the talks; a more systematic and in-depth review of the meeting results will be published in an upcoming issue of EMBOreports. (See also Part II and Part III.)

The talks at this meeting fell into these categories:

Gamma-secretase complex and BACE

Other RIP proteases

AD/PD genetics

Lipoprotein receptor signaling

Tau function

Tau, α-syn, Ab aggregation

With the inaugural talk, Dennis Selkoe of Brigham and Women’s Hospital in Boston, Massachusetts, set the standards by discussing mainly unpublished material. The first part of his talk was about the reconstitution of γ-secretase activity in mammalian cells (see Kimberly et al., 2003). Overexpression of nicastrin, presenilin-1, Aph1 and Pen2 are necessary and likely sufficient to restore γ-secretase activity as measured in a cell-free assay. Coimmunoprecipitations, copurification on an affinity column with immobilized γ-secretase inhibitor, and comigration in glycerol gradients all suggest that the four proteins are in one active complex. A multistep fractionation procedure that involves extraction with one percent DDM and a purification step pulling down FLAG-tagged γ-secretase components after detergent change to one percent digitonin results in sufficiently pure material that can be analyzed in silver-stained SDS-PAGE. The members of the complex are prominently present, but some additional bands are difficult to interpret (contamination or really members of the complex?). Mass spectrometric analysis of these proteins is ongoing.

Dennis Selkoe also elaborated on the AICD (the famous amyloid precursor protein (AβPP) intracellular domain believed to be involved in signaling). This fragment translocates to the nucleus in association with Fe65, and its generation is developmentally regulated in primary neuronal cultures differentiating in vitro. Maximal AICD production is observed when the cultures start to generate synapses. The production inversely correlates with phosphorylation of the intracellular domain of AβPP, specifically Thr-688.
Delta and Jagged, two Notch ligands, can be added to the expanding list of γ-secretase substrates (see ARF related news story). Quite impressive evidence was discussed demonstrating that the liberated Jagged intracellular fragment can reach the nucleus to stimulate the signal from an AP1 reporter assay. The ADAM17/TACE-cleaved, membrane-bound Jagged fragment competes with Notch for cleavage (providing a negative feedback on Notch signaling).
Further work on the Aβ degradation activity of insulin-degrading enzyme (IDE) was shown. Most importantly, IDE-/- mice display decreased Aβ degradation, resulting in 50 percent increase in steady-state levels of Aβ in the brain (see ARF related news story). Interestingly, in these mice, AICD also accumulates (mainly the nonphosphorylated form). Aβ oligomers injected into living anesthetized rats inhibit long-term potentiation in vivo and also in hippocampal slices, and c-Jun N-terminal kinase inhibitors can reverse this effect. In regard to the vaccination studies (see also end of the meeting), it should be noted that the meningoencephalitis-causing T cell response is mainly induced by epitopes in the C-terminal parts of Aβ, while the N-terminal parts are involved in the generation of the (hopefully curative) humoral response.

Finally, a word on Parkinson’s: an O-glycosylated (N-acetylglucosamine and sialic acid-containing) species of α-syn called α-Sp22 has been identified in human brain (see ARF related news story). This α-Sp22 binds to parkin, but not any longer when deglycosylated. α-Sp22 has been purified from brain and was found to be glycosylated on Ser-9 or Thr-22. This raises the question of how this cytoplasmic protein gets glycosylated.

Stefan Lichtenthaler of the Ludwig Maximilians University of Munich, Germany, presented a novel substrate for β-secretase (BACE). He checked different candidate proteins (L-selectin, TNFR2, AβPP and PSGL-1) and found that, of this series, only AβPP and PSGL-1 "shedding" could be increased upon BACE transfection. In BACE1-deficient cells, this processing is extinguished; transfecting BACE restores it. PSGL-1 is P-selectin glycoprotein ligand-1 and mediates leukocyte rolling on the endothelium, allowing transmigration and tissue invasion of leukocytes. Cleavage of PSGL-1 occurs in the juxtamembrane domain.

Patrick Keller of the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, reviewed the many indications for a role of cholesterol in AβPP processing. He went on to discuss the specific association of BACE and a fraction of AβPP with "lipid rafts" (Ehehalt et al. 2003). These are glycosphingolipid subdomains in the cell membrane. YFP-[wt]AβPP and BACE-CFP co-patch with placental alkaline phosphatase, but not with transferrin receptor[?5-41] in such cell surface rafts. Antibody-mediated cross-linking of the rafts increases Aβ production in a cholesterol-dependent manner, probably by fusing rafts and bringing more AβPP and BACE in contact with each other. Some evidence suggests that BACE and its cell surface substrates (such as AβPP) come together during endocytosis by "raft clustering," increasing Aβ production and secretion.

Takeshi Iwatsubo, University of Tokyo, wondered about the individual roles of nicastrin, APH-1, and PEN-2 in the presenilin complex. RNAi knockdown of each individual protein abolished Aβ production in Schneider cells and γ-secretase activity (both with AβPP and Notch substrates) in membrane preparations. Drosophila PEN-2 (dPEN-1) RNAi causes an accumulation of full-length Drosophila presenilin that is stabilized and engaged in a high molecular weight complex. However, co-RNAi of dAPH-1 or dNicastrin abolishes this full-length presenilin accumulation. These effects were also seen in HeLa cells, SY5Y cells, Neuro2a cells and mouse primary neurons. Overexpression of dPEN-2 facilitates the formation of a high-molecular-weight presenilin complex by increasing the accumulation of presenilin fragments, and results in increased γ-secretase activity. Human PEN-2 can rescue RNAi-mediated dPEN-2 deficiency concomitant with conversion of full-length presenilin to fragments. [G112D]dAPH-1 (conserved Gly in TM4) mutation abolishes the stable expression of dNicastrin and dPEN-2 and, consequently, γ-secretase assembly. Overexpression of the three cofactors with presenilin increases the accumulation of active presenilin fragments and γ-secretase activity.
These observations lead Iwatsubo to propose the following model: Presenilin is synthesized as light-molecular-weight species, then assembled in a stable high-molecular-weight complex by binding of APH-1 and nicastrin, which stabilize the holoprotein. Presenilin is finally activated upon endoproteolysis facilitated by PEN-2. PEN-2 itself does not resemble a protease, though this possibility is not entirely excluded. It is, however, more likely that PEN-2 acts as an accessory factor, or that it brings presenilin into a cleavable conformation. Some of this work just appeared in Nature (see Takasugi et al., 2003).

Harald Steiner, Ludwig Maximilians University of Munich, reported that nicastrin RNAi prevents formation of the γ-secretase complex. In stable nicastrin RNAi cells, wild-type nicastrin rescues nicastrin maturation, presenilin fragmentation, PEN-2 and APH-1 stabilization, and γ-secretase activity. Mutational analysis indicates that the whole ectodomain of nicastrin is important. Limited trypsin proteolysis suggests a conformational change of nicastrin upon γ-secretase complex formation (immature nicastrin is trypsin-labile; mature nicastrin is trypsin-resistant). This is not due to glycosylation, because SDS unfolding allows trypsin degradation of mature nicastrin, and prevention of glycosylation by kifunensine does not influence trypsin sensitivity. Thus, conformational compaction of nicastrin accompanies its maturation (see Shirotani et al. 2003).

Steiner pointed out that PEN-2 is an essential component of the high-molecular-weight γ-secretase complex. PEN-2 RNAi prevents the formation of the γ-secretase complex and also decreases nicastrin maturation. Each component of the γ-secretase complex is dependent on the expression of all others.

Steiner also reported reconstitution of the γ-secretase complex in yeast, an organism that does not have endogenous γ-secretase. Coexpression of all four components in yeast is associated with presenilin endoproteolysis and allows the liberation of an AβPP-based reporter construct from the membrane. The presenilin D385A active site mutant abrogates this activity. Thus, coexpression of presenilin, nicastrin, APH-1, and PEN-2 is sufficient for γ-secretase activity; no other components are needed, Steiner said. He reported coimmunoprecipitation experiments confirming that the four components are in contact with each other. The reconstituted presenilin-1 complex has γ-secretase activity; C100 is cleaved into Aβ and AICD, and cleavage produces Aβ38, Aβ40, and Aβ42, as well as AICD50 and AICD51. This work just appeared in Nature Cell Biology (see Edbauer et al., 2003).

Bart De Strooper pointed out that the molecular mass of all four components of the γ-secretase complex is about 220kD, and this molecular mass complex is indeed observed in transfected cells. However, the native complex is twice that size (440kDa), raising the question whether dimeric complexes exist. Could there be multiple presenilin complexes that might have differential, perhaps organ-specific functions? De Strooper noted that systematic Northern analysis revealed that liver and kidney have particularly high expression of all γ-secretase components. Aspartate mutants of PS1 are entirely inactive, although they are accurately incorporated into the presenilin complex (including PEN-2 stabilization and nicastrin maturation), as evidenced by complementation of PS1-/- PS2-/- cells.

Nicastrin glycosylation is presenilin-1 gene dose-dependent, but not needed for γ-secretase activity, as kifunensine prevents nicastrin maturation, but nicastrin still makes it to the cell surface and allows formation of γ-secretase activity (see Herreman et al., 2003).
PS1+/- PS2-/- mice show numerous benign skin tumors, a condition similar to human seborrhoic keratosis that is common in the elderly. Tumors also form when double-knockout mice are rescued with Thy1-driven presenilin-1 (Xia et al., 2001). Loss of presenilin alleles increases the concentration of β-catenin and phospho-β-catenin in keratinocytes and fibroblasts, pointing to the importance of presenilin in Wnt signalling in the skin (Kang et al. 2002). Moreover, adult mice have a severe autoimmune phenotype, including enlarged spleens and salivary glands. There are immunoglobulin deposits in several tissues, and autoimmunoglobulin reactions are detectable in the sera of these mice, but the epitope of auto-Ig is not known. Nevertheless, this is a potentially dangerous side effect that must be monitored in γ-secretase inhibitor trials.

Ralf Baumeister, Ludwig Maximilians University of Munich, reported that the C. elegans presenilin-1 homolog Sel-12 is expressed throughout the worm’s life cycle, while the presenilin-2 homolog hop-1 gradually increases expression in larval stages. Spe-4, a third homolog, is expressed only in the L4 stage.
Sel-12 mutant worms have an egg-laying deficit due to defects of the vulvar smooth musculature. Possibly, cerebral bleeding in PS1-/- embryos is caused by a similar defect of blood vessel musculature. Loss of temperature memory indicates a nervous system defect. The [C60S]sel-12 mutation (corresponding to the Italian [C92S] presenilin-1 mutation in humans) mediates increased Aβ42 production in mammalian cells. Both the egg-laying deficit and the neuronal phenotype can be rescued by human presenilin-1 wild-type and only partially by presenilin-containing clinical mutations. In general, FAD mutations are loss-of-function mutations in C. elegans. Therefore, Baumeister suggested that γ-secretase inhibitors that reduce activity even further could increase the problems of AD. He proposed instead to increase presenilin activity to restore Aβ40 production.

A suppressor screen for sel-12 rescue yielded five candidates that rescue the egg-laying deficit and protruding vulva phenotype with higher than 90 percent penetrance. None of the suppressors work in sel-12/hop-1 double mutants; thus, all suppressors depend on hop-1 expression. Spr-5 strongly derepresses hop-1 expression. Spr-3 and spr-4 encode REST-like zinc finger transcription factors. Spr-5 is homologous to polyamine oxidases, which together with CoREST (spr-1) associate with histone acetylases and with the REST transcription factor, all of which act together in chromatin remodelling. The bottom line is that this mechanism derepresses hop-1 expression, allowing for replacement of sel-12 with hop-1. Does hop-1 take sel-12’s place in the complex? (See also Lakowski et al. 2003.)

Matthew Freeman, of the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, reviewed the rhomboids, a novel family of intramembrane-cleaving (i.e., RIP) proteases. Spitz, a TGFα homolog in Drosophila is cleaved by Rhomboid, a multipass transmembrane protein inside the Golgi membrane. Star is needed for transport of Spitz from the ER, and without Star, cleavage of Spitz does not occur. Rhomboid is an intramembrane serine protease distinct from the aspartyl proteases presenilin and signal peptide peptidase. It works as a single enzyme and not as catalytic subunit of a complex. No precleavage of Spitz is necessary for Rhomboid proteolysis. Interestingly, and in contrast with γ-secretase signaling, the extracellular domain released in the Golgi is secreted into the extracellular milieu and serves as the signaling moiety.

Rhomboids are widespread in the animal kingdom. Seven homologues are known in Drosophila, four in mammals, and two in yeast (Rbd1 and Rbd2). What could be the function of rhomboid in yeast, given that this organism does not depend on intercellular communication? To answer this question, Freeman’s lab made yeast knockout strains, and while the Rbd2-knockout is fine, the Rbd1-less strain grows more slowly, especially on glycerol. This points to a respiratory problem. Tbd1 appears to be a mitochondrial protein, and these organelles had a collapsed morphology in the knockout strain. What could the substrate be? In a clever in silico approach, Freeman looked for candidate proteins that met these criteria: mitochondrial, single transmembrane domain, and biochemically characterized as soluble proteins. Candidates included Ccp1p, which is involved in oxidative stress, but its knockout has no consequences, and Mgm1p, a dynamin-like GTPase regulating membrane remodeling. Remarkably, the Mgm1 knockout yields a similar phenotype to the Rbd1 knockout. Further work confirmed that Rbd1p cleaves Mgm1, and mutation of the Mgm1p cleavage site causes the same phenotype (mitochondrial collapse) as the complete deletion of the protein.

Rhomboid is a highly specific protease, cleaving Spitz, but not EGFR, Delta, or TGN38. Even the human homolog of Spitz (TGFα) is not cleaved because it lacks the proper cleavage sequence in the transmembrane domain. Spitz/TGFα swapping revealed that a GA motif around amino acid 140 accounts for this remarkable sequence specificity of rhomboid. The transmembrane helical ASIASGAMCAL sequence is characterized by small and helix-destabilizing amino acids. This sequence was found (manually!) in 20-30 candidates in the mouse genome. Incidentally, the micronemal (i.e., infection-promoting) adhesion proteins in Toxoplasma gondii have similar transmembrane domains and are also subject to intramembrane cleavage. By analogy, rhomboid was found to cleave malaria-relevant MIC proteins. Both findings could yield therapeutically relevant approaches. Thus, control of intramembrane proteolytic cleavage becomes a major issue in many diseases other than AD, as well.

Bruno Martoglio, Swiss Federal Institute of Technology, Zurich, presented an overview of the biology of signal peptide peptidase (SPPase), a presenilin-related intramembrane protein-cleaving protease (see ARF related news story). SPPase is responsible for the generation of the HLA-E epitopes that are generated by cleavage of the signal peptides of certain type II proteins. In the cytosol, a proteolytic activity that remains unidentified (or at least was not discussed at the meeting) chops up the cytosolic fragment. The question arose whether a similar process could be responsible for trimming of the Aβ peptide after release.

Overall, there are many differences between presenilin and SPPase, which is a member of the presenilin homolog family. SPPase has an opposite orientation, is not incorporated in a multimembrane complex, is not activated by presenilinase, and recognizes type II proteins as substrates. Nevertheless, several inhibitors that inhibit presenilin also inhibit SPPase. For instance Merck’s L685,458 inhibitor binds not only to PS but also to SPPase in cross-linking studies. The question arose why this was not noticed in the original publications (see also Weihofen et al., 2003).

Steve Younkin of the Mayo Clinic in Jacksonville, Florida, elaborated on the four candidate AD genes on chromosome 10: PLAU, VR22, IDE, and CYP46. He discussed systematically the evidence in favor of each gene and made a case for pooling the results from genetic studies to demonstrate the significance of the association with late-onset AD. For the four genes, evidence was brought forward for association, even though such association could not be confirmed in John Hardy’s series. This initiated a philosophical discussion on "whether the pint is half-full or half-empty," a problem that was more deeply plumbed in the bar session ("more work is needed"). In any event, PLAU (urokinase-type plasminogen activator)-deficient mice demonstrate an age-dependent increase of plasma Aβ levels. VR22 (α-T-catenin) variants are significantly associated with plasma Aβ42 and the VR22 4360/4783 appears to account for a substantial proportion of chromosome 10 linkage (see ARF related news story). Finally, Aβ is increased in IDE-/- mice and IDE variants are significantly associated with plasma Aβ42 in some studies. Cholesterol-24-hydroxylase (CYP46) significantly associates with AD in a large multicenter study of almost 600 AD vs. 600 controls (see ARF related news story).

John Hardy of the National Institute on Aging in Bethesda, Maryland, mentioned that the chromosome 10 locus appears to contain multiple AD genes. Tested candidates include IDE, PLAU, and VP22, but all turned out negative in three series (Cardiff, Washington University, Mayo Clinic Jacksonville) of more than 300 cases and 300 controls. Apparently, there is a difference of opinion between the Younkin and the Hardy consortium. The prior discussion on the state of the pint came up again, and Hardy concluded the topic with a typical one-liner: "What’s confirmed by John Hardy is fact."

As for Parkinson’s genes, pseudo-dominant inheritance of the parkin phenotype was observed in no less than 10 heterozygote mutations that segregate with disease, pointing to haploinsufficiency. Two SNPs in the parkin promoter segregate with disease and affect promoter activity; these are especially frequent in single-mutation parkin patients (these could be the cases with Lewy bodies). α-syn haplotypes are associated with Parkinson’s. α-syn overexpression, especially its mutant forms, inhibits proteasomal activity in M17 cells and enhances toxicity of proteasome inhibitors, while wild-type parkin overexpression ameliorates this. Upon transfection, a quite specific toxicity of A53T α-syn to tyrosine hydroxylase-positive neurons in ventral mesencephalic neuron cultures is observed.

Finally, the PARK7 locus was rigorously linked to the DJ-1 gene by the identification of about 10 deletion and nonsense mutations (see ARF related news story). When asked what he thought about the involvement of DJ-1 in the cellular handling of oxidative stress, Hardy drew laughter with his second deadpan reply: "Oxidative stress is like apple pie."

Joachim Herz, University of Texas Southwestern Medical Center, Dallas, discussed the mechanisms of signaling by the lipoprotein receptors: LDLR, VLDLR, MEGF7, ApoER2, LRP, LRP1B and megalin. ApoE receptors mediate signal transduction in neurons. ApoE receptors (VLDLR and ApoER2) control brain development. Reelin usually binds to these two receptors and stimulates tyrosine phosphorylation of the adaptor protein disabled (Dab1), activating a cascade from PI3K to Akt to GSK3β to tau hyperphosphorylation (see ARF related news story). Reelin mice, as well as VLDLR- and ApoER2-knockout mice, show increased tau phosphorylation. Dab1-/- mice do, also, but the extent of this is dependent on the genetic background. For example, 129xC57BL6 is permissive, while little or no tau phosphorylation is apparent in the Balb/c. The reelin mutation is viable in this background, as well. Microsatellite analysis of F1 intercrosses of the two strains reveals quantitative trait loci (QTLs). Interestingly, peaks of association were found on chromosome 1, 12, and a strong one on chromosome 16; PS1 and AβPP reside in the QTLs on chromosome 16 and chromosome 12, respectively.

Finally, Herz provided nice insights into novel functions of the ApoE receptors in signaling. PDGF-BB induces tyrosine phosphorylation of LRP via caveolar PDGF receptors, allowing binding of the adaptor protein SHC. This is prevented by ApoE lipoproteins and by the tyrosine kinase inhibitor Gleevec, now used in cancer therapy. Smooth-muscle cell-specific LRP-knockout mice on a high cholesterol diet show stunning atherosclerosis and aortic aneurysms. A Gleevec-laced diet rescues this phenotype. LRP-deficient aortas show an enormous increase in PDGF-R signaling. Thus, this pathway could contribute importantly to the atherosclerotic phenotype.

EM Mandelkow

Eva-Maria Mandelkow, of the Max-Planck Institute for Structural and Molecular Biology in Hamburg, provided interesting information on the pathway of how tau stabilizes microtubules and mediates axonal transport. In AD, tau disassembles from microtubules (MTs) and forms paired helical filaments (PHFs), but it is unclear whether these are directly toxic to the neuron. It is possible that MT-bound tau can be harmful, too. Proline-directed phosphorylation yields epitopes that are recognized by the most commonly used AD-diagnostic antibodies, but these phosphorylations barely affect tau binding to MTs. MT disassembly occurs after phosphorylation of the C-terminal KXGS motifs catalyzed by MARK kinases, for example. In fact, MARK2 induces neurite outgrowth, and dominant-negative forms of MARK2 inhibit neurite outgrowth in N2A cells (Biernat et al., 2002). Interestingly, MARK2 tagged with a HA epitope co-localizes with F-actin, and phospho-tau (12E8) localizes to phalloidin-positive actin filaments. Physiologically, KXGS site phosphorylation allows for dynamic microtubules as exist in growth cones, for example.

What, then, is the effect of increased binding of tau to MT? Apparently, tau inhibits plus-end directed transport by competing with kinesin for the same binding site on MT. When tau is overexpressed, more of it binds to MTs, increasingly hindering both antero- and retrograde transport. Mandelkow measured this by determining the run length and velocity of individual vesicles. The velocity does not change, but the run length is shortened in both directions by tau. Since tau also interferes with the binding of kinesin, but not dynein, to MT, its net effect is that retrograde transport becomes dominant. MARK influences this process by causing the removal of the tau obstacle. In conclusion, tau inhibits and MARK facilitates the transport of active mitochondria, influencing synaptic energy production. Tau40 overexpression also inhibits the transport of AβPP vesicles. Tau thus leads to accumulation in the cell body of axonal transport cargoes (synaptic vesicles, mitochondria, etc.) with imaginable adverse effects including, for example, increased sensitivity to H2O2.

Continuing along similar lines, Eckhard Mandelkow stated that tau is a natively unfolded and highly soluble protein, whose fibril formation accelerates in the presence of additional factors such as polyanions (heparin, polyglutamine, DNA). Indeed, phosphorylation prevents aggregation, and it is therefore surprising that PHF can form despite tau being phosphorylated. The VQIVYK sequence in the tau protein tends to form β-structures, and it associates with this motif in other tau proteins to nucleate further assembly, leading to classical amyloid. The FTDP-17 mutations have little effect on MT stability. Instead, P301L and _K280 FTDP-17 mutations in tau accelerate PHF assembly, perhaps by favoring β-strand conformation
Tryptophan mutation scans and autofluorescence measurements performed to analyze the vicinity of domains within PHFs reveal that the hexapeptide motifs that form the aggregation nucleus are buried in the PHFs. Interestingly, PHFs in vitro have low intrinsic stability; they are easily denatured by guanidine as monitored by tryptophan fluorescence. Screening of PHF inhibitor compounds is based on thioflavin S fluorescence. Actually, tau bound to MT induces thioflavin S fluorescence like in PHFs, and overloading of tau on MTs leads to some filament-like structures on the surface of microtubules. (See also Barghorn et al., 2002.)

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References

News Citations

Paper Citations

Kimberly WT, LaVoie MJ, Ostaszewski BL, Ye W, Wolfe MS, Selkoe DJ. Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc Natl Acad Sci U S A. 2003 May 27;100(11):6382-7. PubMed.
Ehehalt R, Keller P, Haass C, Thiele C, Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol. 2003 Jan 6;160(1):113-23. PubMed.
Takasugi N, Tomita T, Hayashi I, Tsuruoka M, Niimura M, Takahashi Y, Thinakaran G, Iwatsubo T. The role of presenilin cofactors in the gamma-secretase complex. Nature. 2003 Mar 27;422(6930):438-41. PubMed.
Shirotani K, Edbauer D, Capell A, Schmitz J, Steiner H, Haass C. Gamma-secretase activity is associated with a conformational change of nicastrin. J Biol Chem. 2003 May 9;278(19):16474-7. PubMed.
Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Reconstitution of gamma-secretase activity. Nat Cell Biol. 2003 May;5(5):486-8. PubMed.
Herreman A, Van Gassen G, Bentahir M, Nyabi O, Craessaerts K, Mueller U, Annaert W, De Strooper B. gamma-Secretase activity requires the presenilin-dependent trafficking of nicastrin through the Golgi apparatus but not its complex glycosylation. J Cell Sci. 2003 Mar 15;116(Pt 6):1127-36. PubMed.
Xia X, Qian S, Soriano S, Wu Y, Fletcher AM, Wang XJ, Koo EH, Wu X, Zheng H. Loss of presenilin 1 is associated with enhanced beta-catenin signaling and skin tumorigenesis. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10863-8. PubMed.
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Lakowski B, Eimer S, Göbel C, Böttcher A, Wagler B, Baumeister R. Two suppressors of sel-12 encode C2H2 zinc-finger proteins that regulate presenilin transcription in Caenorhabditis elegans. Development. 2003 May;130(10):2117-28. PubMed.
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Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part II

11 Apr 2003

The talks at this meeting fell into these categories:

Fibril formation
Tauopathies, α-synucleinopaties
α-synuclein function
Parkin, synphilin, ubiquitination

Hilal Lashuel of Brigham and Women's’ Hospital in Boston, Massachusetts, talked about the biophysics of fibril formation. A cascade of folded protein to protofibril (oligomers) to protofilaments (thin) to fibrils (cross-β-sheet amyloid) is postulated, and toxicity could increase along this cascade. The working hypothesis is that the protofibrils, rather than the fibrils, are the toxic species of amyloids in general. Synthetic α-synuclein (αSYN) protofibrils display some heterogeneity.

Investigation of the various protofibrillar species shows that some of them resemble pore-like tubes that allow passage of small molecules ([E22G]Aβ), IAPP (type II diabetes), serum amyloid A (uniform hexameric channels), prion protein, and [A4V]SOD1 (see ARF related news story); Lansbury interview; Wang et al., 2002; Lashuel et al., 2002; Lashuel et al., 2002b). Remarkably, when wild-type and arctic Aβ are mixed, the amount of protofibrils increases.

Virginia Lee, University of Pennsylvania School of Medicine, Philadelphia, started with a summary of the three classes of FTDP-17 tau mutations:

mutations impairing tau function (reduced MT binding) G272V
mutations promoting tau aggregation P301L
mutations altering exon 10 splicing N272N

Although tau is predominantly a neuronal (i.e., axonal) protein, numerous glial tau inclusions exist, as well: astrocytic plaques, tufted astrocytes, astrocytic inclusions, oligodendrocytic inclusions (coiled bodies), and neuropil threads in corticobasal neurodegeneration (CBD) white matter. There are many tauopathies, both primary ones (FTDP-17, CBD, Pick’s disease) and secondary ones (where tangles develop as a response to a primary insult). Lewy bodies are intraneuronal inclusions consisting of straight filaments of α-syn. Additional syn family members exist: β-syn lacks repeat 6, which is responsible for fibril formation of α-syn, and γ-syn is expressed in peripheral neurons. α-syn mutations (A30P and A53T) cause familial PD. Like with tau, there are many synopathies, including Parkinson’s, dementia with Lewy bodies (DLB), Lewy-body variant of Alzheimer’s (LBVAD), neurodegeneration with brain iron accumulation type 1 (or Hallervorden-Spatz disease), pure autonomic failure, and multiple system atrophy (MSA).

Both tau and α-syn are abundant, small, highly soluble, amphipatic neuronal proteins, with a long half-life and the potential to become pathologically phosphorylated. Both aggregate into 10-20nm filaments, generate true amyloid and can be ubiquitinated, nitrated and phosphorylated. Remarkably, both co-occur in LBVAD, AD, familial AD, Down’s syndrome, but also in diseases with scant Aβ pathology: in DLB and PD there is partial overlap of immunofluorescence within cell bodies and neurites. In FTPD-17, there is co-localization of tau and α-syn in glial cytoplasmic inclusions of MSA. α-syn and all six isoforms of tau synergistically enhance each other’s fibrillization in vitro, as detected by K114 fluorometry. Interestingly, homopolymers are formed, so there is synergistic formation of separate α-syn and tau fibrils. In (PrP)-[A53T] α-syn mice, there is tau pathology in brainstem and spinal cord. Bigenic mice expressing tau and α-syn under control of the oligodendrocyte-specific CNP promoter have enhanced fibrillization of tau and α-synin white matter. Also, in an A53T α-syn mutant patient, a lot of tau pathology occurs even in regions where little α-syn is observed.

John Trojanowski, University of Pennsylvania School of Medicine, Philadelphia, discussed the possible functions of α-syn, which include:

vesicle binding
regulation of the number of synaptic vesicles
synaptic transmission
chaperoning
phospholipase D2 inhibition

Epitope mapping with many α-syn monoclonal antibodies revealed the presence of full-length α-syn in all α-synopathy lesions. High molecular weight α-syn species can be extracted from patient brains. In glial cytoplasmic inclusions of MSA, C-terminal α-syn epitopes are immunodominant, whereas in all the other lesions the epitopes are equally represented. α-syn in lesions is ubiquitinated, nitrated, and phosphorylated. Oxidized α-syn antibodies reveal striatal pathology. HSP70 rescues dopaminergic neurons from degeneration in the α-syn fly model (see ARF related news story). Indeed, α-syn lesions contain HSP70 and HSP40.

Mice highly overexpressing h[A53T] α-syn driven by the PrP promoter develop progressive locomotor deterioration. Age of onset is between nine and 16 months, and the phenotype is lethal within a month of outbreak. The sites of α-synopathy are outside of the dopaminergic system. Insoluble α-syn filaments accumulate in the transgenic mice. For more on α-syn mouse models, see ARF related news story. Finally, posttraumatic recovery is impaired in a neurofilament-inclusion-bearing mouse model, because of increased necrotic neuron loss. Perhaps the induction of iNOS in the traumatic area contributes to this sensitization.

Philipp Kahle, Ludwig Maximilians University of Munich, centered his presentation around a recently recognized pathological feature of α-syn, namely proteinase K (PK) resistance (Neumann et al., 2002). A parallel study was undertaken with postmortem brains that were split, one hemisphere frozen and the other fixed in formalin. PK resistance of α-syn was assessed in biochemically isolated fibrils and in situ on digested paraffin-embedded tissue blots. This revealed classical α-synopathy, as well as previously underappreciated pathology. Generally, the regional distribution of PK-resistant α-syn correlated with the stage of α-synopathy, suggesting that Lewy pathology constitutes a primary lesion in the classical disease course, which spreads from the medulla to the brainstem, and then to the limbic and neocortical systems. Interestingly, FAD patients displayed strong α-synopathy in the amygdala, indicating that under certain circumstances, and in the limbic system specifically, α-syn may fibrillize secondary to AD plaque deposition. Kahle went on to point out that the A30P mutation of α-syn also causes Lewy pathology and locomotor deterioration in a transgenic mouse model. As in human patients, PK-resistant and Ser-129-hyperphosphorylated α-syn fibrils developed in phenotypic transgenic mice. Additional markers of α-synopathy in the A30P α-syn mice (Thy1 promoter) included silver-positive dystrophic neurites, thioflavin S-positive Lewy bodies and Lewy neurites, electron-dense fibrils clogging swollen neurites, oxidative protein modifications, and gliosis, and ubiquitination of the lesions. Interestingly, the sites of α-syn fibril formation were in the spinal cord, brainstem, and deep mesencephalic nuclei, but the nigrostriatal dopamine system remained unaffected.

In a second talk, Iwatsubo reported on the purification of 3.5 million Lewy bodies (LB) to generate the LB-specific monoclonal antibody LB509. MALDI-TOF analysis of LB-derived α-syn revealed Ser-129 phosphorylation, whereas soluble α-syn was not phosphorylated. Also, mono- and diubiquitinated α-syn was identified. These are being purified by size-exclusion HPLC. Iwatsubo used the Mec-7 promotor to drive α-syn expression touch neurons of C. elegans, but even aged animals of this strain did not hyperphosphorylate α-syn. The touch response is impaired in transgenic worms expressing the PD-causing A30P mutant of α-syn, but less so in worms expressing the other mutant A53T; almost no deficiency was seen with wild-type α-syn. Using the dat1 promoter, α-syn was expressed in dopaminergic neurons. In these cells, α-syn sometimes becomes hyperphosphorylated. The function of dopaminergic neurons in live C. elegans can be evaluated by measuring a decrease in body bending and movement in food-rich areas. Indeed, A53T and A30P α-syn expressed under control of the dat1 promoter did decrease the worm’s typical motor behavior in food-rich areas; adding L-DOPA rescues this phenotype. Baumeister cautioned that the arrest of body bending and movement in food-rich areas by α-syn overexpression in C. elegans may be unspecific, since β-syn expression, for example, produces the same phenotype.

Ryosuke Takahashi of the RIKEN Brain Science Institute in Saitama, Japan, spoke about the autosomal recessive form of juvenile parkinsonism caused by mutations in parkin. Parkin has E3 ubiquitin ligase activity and mutant parkin shows enzymatic deficiency in vitro. One of the substrates of parkin is Pael (Parkin-associated endothelin receptor-like) receptor (see ARF related news story). This protein is almost exclusively expressed in the dopaminergic neurons of the substantia nigra. In the rest of the CNS, Pael-R is mostly localized in oligodendrocytes. Parkin regulates Pael-R turnover in the context of endoplasmic reticulum-associated degradation. Proteasome inhibition by six-hour lactacystin treatment causes Pael-R accumulation in the ER of SH-SY5Y cells; longer treatment leads to Pael-R aggresome formation. Unfolded (more specifically, insoluble) Pael-R accumulates in AR-JP (but not idiopathic PD) brain, but no Pael-R aggregates are stained in patient brain. Transgenic Drosophila expressing Pael-R show selective degeneration of DA neurons (Yang et al., 2003).

Other parkin-binding proteins are HSP70 and CHIP (C-terminus of Hsc70-interacting protein). CHIP contains a C-terminal TPR domain (Hsp70/Hsp40 binding) and a U box (E2 binding). It is a cofactor of the parkin complex that enhances parkin’s E3 activity, particularly when CHIP and Hsp70 are upregulated by ER stress (mediated by tunicamycin). Hsp70/Hsp40 induction peaks six hours after tunicamycin addition (initially preventing Pael-R aggregation), and then CHIP gradually increases for 24 hours, helping parkin to degrade the accumulating, misfolded Pael-R.

Christopher Ross of Johns Hopkins University School of Medicine, Baltimore, Maryland, reviewed the aggregation-promoting properties of the α-syn-interacting protein synphilin (SPH-1), and demonstrated that parkin interacts with synphilin-1 (see ARF related news story). Parkin ubiquitinates synphilin-1 in the cytosolic inclusions formed in cells cotransfected with SPH-1 and α-syn. α-syn was not a substrate for parkin, but co-localized with SPH-1 and parkin in the inclusions. Interestingly, parkin-mediated ubiquitination did not reduce the half-life of SPH-1, because it catalyzed poly-ubiquitination via K63 rather than K48. Unlike K48-linked polyubiquitin chains that direct the attached proteins to proteasomal degradation, K63-linked ubiquitination influences subcellular localization or signaling of the target proteins. It is of prime importance to clarify the biology and potential relevance to Parkinson’s of K63-linked polyubiquitination catalyzed by parkin.

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References

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Wang L, Lashuel HA, Walz T, Colon W. Murine apolipoprotein serum amyloid A in solution forms a hexamer containing a central channel. Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15947-52. PubMed.
Lashuel HA, Petre BM, Wall J, Simon M, Nowak RJ, Walz T, Lansbury PT. Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils. J Mol Biol. 2002 Oct 4;322(5):1089-102. PubMed.
Neumann M, Kahle PJ, Giasson BI, Ozmen L, Borroni E, Spooren W, Müller V, Odoy S, Fujiwara H, Hasegawa M, Iwatsubo T, Trojanowski JQ, Kretzschmar HA, Haass C. Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies. J Clin Invest. 2002 Nov;110(10):1429-39. PubMed.
Yang Y, Nishimura I, Imai Y, Takahashi R, Lu B. Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila. Neuron. 2003 Mar 27;37(6):911-24. PubMed.

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Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part III

11 Apr 2003

The talks at this meeting fell into these categories:

β-syn, Lentivirus
GDNF
Immunotherapy and CAA update
Secretase inhibitor update
NSAID update
Statin update
β-sheet breakers

Eliezer Masliah of the University of California, San Diego, discussed the interplay of syn family members in human patients and transgenic mouse models. β-syn is expressed at even higher levels than α-syn in the brain, and the mRNA levels of syn family members differ from controls in AD (relatively more γ-syn) and LBD (relatively more α-syn). β-syn prevented α-syn aggregation in vitro even under oxidative conditions, and lentiviral delivery of β-syn decreased α-syn aggregation in cell culture. Moreover, β-syn suppressed α-syn aggregation in vivo: Crossbreeding with β-syn transgenic mice and lentiviral delivery of β-syn both reduced protein aggregation in α-syn transgenic mice, and synapse loss was ameliorated. Conversely, AD pathology appeared to exacerbate α-synopathy in α-syn transgenic mice: Crossbreeding with AβPP transgenic mice that produce excessive Aβ42 exacerbated α-syn aggregation and phenotype, possibly involving oxidative stress conferred by LRP-mediated Aβ42 uptake. Interestingly, Aβ40 suppressed α-syn aggregation. (see ARF related news story).

Patrick Aebischer, Lausanne University Medical School, Switzerland, gave a superb overview of the potential for lentiviral gene delivery systems to generate animal models and therapeutic approaches for neurodegenerative diseases. First, dominant genes (α-syn, Htt, SOD1, etc.) could be silenced by application of siRNA-coding lentivirus (see ARF live discussion). Proof of principle was achieved for viral siRNA knockdown of GFP in cell lines and silencing of SOD1 in a transgenic mouse model of ALS. Second, expression of recessive genes (parkin, DJ-1, etc.) could be restored. Third, neuroprotective and neurotrophic genes could be delivered. For example, lenti-GDNF injected into the substantia nigra of MPTP-intoxicated primates caused impressive behavioural improvement, total rescue of 18fluoro-DOPA PET, and fiber sprouting. Aebischer noted that it might even become possible to satisfy individual demands of therapeutic doses of GDNF by the use of tetracycline-regulatable lentiviral constructs.

Deniz Kirik of Lund University, Sweden, explained that acute dopamine neurotoxin models are extremely effective, but too rapid for useful neuroprotection studies. In a milder rat model based on intracranial injection of low-dose 6-hydroxydopamine, the neurotrophic factor GDNF was found to be neuroprotective when delivered repeatedly to terminals during the degenerative process, it even protected the rats against locomotor deterioration. First, clinical trials showed that intrastriatal infusion of GDNF alleviated symptoms in five Parkinson’s patients to an extent similar to standard L-DOPA monotherapy (see ARF related news story). Kirik went on to describe the phenotypes of rat and monkey models of Parkinson’s based on adeno-associated viral gene delivery of α-syn, which recapitulate early α-synopathy and reversible, selective dopamine neuron loss (see ARF related news story).

Dale Schenk of Elan Pharmaceuticals, South San Francisco, California, reviewed approaches to Aβ immunotherapy in three categories: active immunization with Aβ, active immunization with immunoconjugates (Aβ fragments conjugated to an ovalbumin-derived T cell antigen), and passive immunization with purified monoclonal antibodies against Aβ.

All three approaches reduce Aβ burden in transgenic mouse models. The first caused a dramatic reduction of plaque burden and gliosis in two different transgenic mouse models, as well as cognitive improvement. Cross-reactivity with soluble Aβ was observed, but capture of soluble Aβ did not correlate with therapeutic efficacy.

Optimizing Aβ immunotherapy requires knowing the most effective epitopes and immunoglobulin class. AN 1792 immunization yields antibodies against epitopes mostly in the N-terminus of Aβ in mice and humans. IgG2a (2C1 and 12B4) immunoglobulins proved the most effective subtype for clearing Aβ and reducing the neuritic burden in passively immunized mice, probably because this antibody subtype interacts most efficiently with Fc receptors (see ARF related news story).

Schenk also discussed two potential side effects of Aβ immunotherapy. Transgenic mice with a high burden of vascular amyloid suffer cerebral hemorrhage after passive immunization with a monoclonal IgG1 directed against an N-terminal Aβ epitope (see Mathias Jucker’s talk). T cell invasion into brains of C57Bl/6 mice was observed upon AN 1792 immunization plus concomitant pertussis toxin treatment (see ARF related news story). Both efficacy and side effects were investigated in the first autopsy study of an AN 1792-treated patient (see ARF related news story). In this patient, Aβ immunotherapy was effective as evidenced by large plaque-free areas in the temporal cortex. Aβ-phagocytosing microglia were visible. This patient suffered excessive inflammation in the brain that was recognized in an MRI scan and controlled by dexamethasone treatment, though she never fully recovered. She died of a pulmonary embolism 12 months after the last dose. Despite the marked reduction of senile plaques in this immunized patient, neurofibrillary tangles and cerebrovascular amyloid persisted in the disease course of this person.

Christoph Hock, University of Zurich, continued the theme by reporting on the Zurich cohort (30 Aricept-treated AD patients) of the immunization trial (see ARF related news story). Immunized patients developed strong and sustained anti-Aβ immune sera. The human antisera intensely and selectively stained compact, diffuse, vascular plaques but displayed no cross-reactivity with soluble Aβ. Such antibodies were also collected from some immunized patients’ CSF. Interestingly, the antibody titers did not correlate with staining intensity, suggesting distinct avidity of the antibodies in each patient. Antibody titers also did not correlate with the incidence of aseptic meningoencephalitis, which could be treated with methylprednisolone. Publication of the first report on cognitive amelioration of the immunized AD patients can be expected by the end of this year.

Mathias Jucker, University of Basel, Switzerland, explained his hypothesis of the mechanism of vascular amyloidosis derived from transgenic mouse models. Expression of AβPP exclusively in neurons of (Thy1)-AβPP mice bred into an AβPP null background causes cerebral amyloid angiopathy to develop, demonstrating that neuronal expression of AβPP is sufficient. Blood levels of Aβ are low in this mouse model, so vascular amyloid is unlikely to be blood-borne. An AβPP dutch mouse model of hereditary cerebral hemorrhage with amyloidosis reveals that all the amyloid is in the cortical vasculature. Upon aging, these mice have hemorrhages and neuroinflammation. AβPPdutch mice show very little 42-ending Dutch Aβ, but this can be induced upon crossbreeding with PS1G384A. In conclusion, high Aβ42 levels lead to plaque formation in the neuropil, whereas high Aβ40 levels allow amyloid transport along the perivascular drainage pathway, where vascular amyloid eventually builds up.

In AβPP23 mice, passive immunization with β1 monoclonal IgG1 against Aβ (3-6) mainly decreases diffuse plaques and Aβ42 levels. Immunotherapy has no beneficial effect on CAA; indeed, these mice have significant cerebral hemorrhage, possibly because of the enormous burden of vascular amyloid in this particular mouse model (see ARF related news story). Under these circumstances, decreases in cerebral blood flow were evident by functional MRI on anesthetized mice. Finally, Jucker cautioned that although EM analysis reveals microglia in close proximity to amyloid, Aβ was never visualized inside microglia. Moreover, the "sink hypothesis" of decreased Aβ levels leading to plaque removal is still unproven. Thus, the mechanism of plaque clearance upon Aβ immunotherapy remains to be solved (see also ARF related news story).

Martin Citron of Amgen Inc, Thousand Oaks, California, talked about BACE1, the enzyme conferring β-secretase activity. Because β-secretase cleavage is the first step in Aβ production, and BACE is elevated in AD cortex (see ARF related news story), it is a prime target for antiamyloidogenic drug development. Peptidomimetic BACE1 inhibitors are published, but small-molecule BACE inhibitor development is difficult. Early attempts with inhibitors developed against proteases with sequence similarity (e.g., HIV protease) were disappointing. Solving the crystal structure of BACE1 revealed a highly complex active center with no less than eight subdomains, possibly inspiring rational drug design.

What side effects might be expected from therapeutic BACE inhibition? In addition to AβPP, there are additional substrates (ST6gal I, PSGL-1, and probably more) that could be vitally dependent on BACE cleavage. Nevertheless, knocking out BACE1 in mice caused no phenotype, even upon aging. Even though these mice have neither β-secretase nor Aβ, they have no pathology and no changes in gene expression as measured by Affimetrix mouse chips. Proof of concept for an antiamyloidogenic effect of BACE1 inhibition was provided by the finding that crossing Tg2576 with BACE1-/- mice suppressed plaque formation in aged offspring.

Mark Shearman, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom, described a novel series of small-molecule γ-secretase inhibitors. WO 0236555 is the lead compound of sulfonamido-substituted, bridged bicycloalkyl derivatives that decrease plasma Aβ, soluble brain Aβ, and AβPP CTFs. Screening for selective AβPPγ (42) cleavage inhibition provided evidence that certain NSAIDS (ibuprofen, flurbiprofen, sulindac, etc.) act as noncompetitive γ-secretase inhibitors. These compounds also inhibit Notch cleavage at high concentrations. In fact, 60 compounds compared for AβPP cleavage and Notch cleavage revealed no differential efficacy at all. This cross-reactivity of γ-secretase inhibitors points to the potential side effect of altered immune responses due to blocked Notch-dependent hematopoiesis (i.e., T cell development).

Edward Koo, University of California, San Diego, reported that ibuprofen lowers Aβ42 production independent of cyclo-oxigenase (COX) inhibition, because the Aβ42-lowering properties are maintained in fibroblasts from COX-/- mice. Not all NSAIDs reduce Aβ42. Some, for example, celecoxib, actually increase Aβ42 with a reciprocal reduction of Aβ38, while others, for example, sulindac, lower Aβ42 production concomitant with an increase of Aβ38. In fact, among the NSAIDs clinically assessed for AD to date, indomethacin was the only one that ameliorated AD symptoms, and it was also the only one with Aβ-lowering properties. It will be interesting to optimize drugs for a maximal Aβ42-lowering effect and eliminated COX cross-reactivity. A two-center phase I trial with R-flurbiprofen in 48 healthy elderly (55-80y) subjects just started, assessing safety, tolerability, pharmacokinetics, and blood- and CSF-Aβ biomarker effects in AD patients.

The molecular mechanism of the Aβ42-lowering effect of some NSAIDs is not clear. AID/AICD generation or stabilization by Fe65 is apparently not decreased, arguing against direct γ-secretase inhibition. However, in CHO cells cotransfected with FAD presenilin mutants, NSAIDs generally have a stronger Aβ42-lowering effect (with the notable exception of _ex9 presenilin-1). Perhaps certain NSAIDs exert allosteric γ-secretase modulation. (see related NSAID coverage; NSAID live chat).

Mika Simons of the University of Tübingen, Germany, mentioned that cholesterol depletion leads to an 80-90 percent reduction of Aβ production, while adding back cholesterol restores Aβ levels. This is due to a cholesterol-mediated decrease of α-secretase and increase of β-secretase activity. The reason for this effect may be correlated to the amount of AβPP in rafts, where β-secretase cleavage might occur. In brain there is no cholesterol uptake from LDL, rather it must be synthesized de novo. Thus, BBB-permeant statins like simvastatin could selectively lower intracellular cholesterol levels in neurons. Indeed, treatment of guinea pigs with simvastatin lowers brain Aβ levels with minimal side effects (e.g., changes in liver enzyme levels); all effects are reversible upon simvastatin washout in the guinea pigs.

Retrospective metastudies indicate that hypertensive patients treated with statins had a lower prevalence of AD. Prospective studies of statin treatment against coronary diseases and stroke with cognitive monitoring were inconclusive, so AD-specific studies are warranted. The first clinical trial of simvastatin in mild-moderate AD patients proved somewhat efficacious (Simons et al., 2002). Plasma LDL cholesterol decreased by 50 percent, CSF free cholesterol decreased by 10 percent. MMSE scores improved slightly in mild cases only, and this correlated with decreased cholesterol levels. No beneficial effect was observed in moderately impaired AD patients, perhaps the plaque load may have already been too high. CSF tau levels were not decreased.

Claudio Soto at Serono Pharmaceutical Research Institute in Plan-les-Ouates, Switzerland, first summarized general issues of protein misfolding disorders. They include intracellular vs. extracellular inclusions, peptide-specific vs. generalized inclusions, the deposition of misfolded protein aggregates vs. misfolding of soluble (dysfunctional) protein, loss-of-function vs. gain-of-toxic-function, and the involvement of posttranslational modifications.

Soto talked about developing β-sheet breakers based on peptides that intercalate with the amyloid-seeding peptide sequence of aggregation-prone proteins spiked with conformation-breaking proline residue(s). In the case of Aβ, such an anti-aggregation peptide specifically prevents Aβ (but not prion or amylin) fibrillization in vitro as well as in AβPPld-transgenic mice. Administration of the β-sheet breaker ameliorated the astrogliosis and microglial activation, as well as the minor neurodegeneration normally observed in this mouse model. β-Sheet breakers led to improvement in water maze performance in a rat model of amyloidosis caused by Aβ injection, but there was only partial reduction of plaque burden. The groups’ current focus is on the development of Aβ-sheet breakers with improved pharmacokinetics. Soto said that the toxicology so far is encouraging and no immunological responses to β-sheet breaking peptides were seen. Compound optimization strategies include the development of small peptidomimetics and nonhydrolysable peptide derivatives. Peptidomimetic approaches are being pursued for various different indications but have not, to date, generated novel drugs, as technical challenges remain.

Leon Thal, also at University of California, San Diego, concluded the program by reviewing the clinical course of AD with a specific emphasis on the design of prospective clinical trials. He pointed out that the study end-points must be carefully selected and followed through. More importantly, patients must be recruited early in the disease, ideally right around the first manifestation of mild cognitive impairment, or even prior to symptoms. He also noted that although AD varies considerably from patient to patient, the progression of this disease is rather uniform and predictable, even in limited cohort sizes.

This was a great meeting that brought together interesting people who presented excellent science with great enthusiasm. Never has it been clearer that improved therapy is within reach for Alzheimer’s disease, and that improvements are underway for Parkinson’s, as well. The enchanting scenery of the locale served as an allegory for the hopeful vista we received toward the future.—Philipp Kahle, Ludwig Maximilians University of Munich, Germany, and Bart De Strooper, Flanders Interuniversity Institute for Biotechnology and KU Leuven, Belgium.

International Titisee Conference 2003

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Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part I

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Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part II

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Philipp Kahle and Bart De Strooper Report from Lake Titisee, Germany: Part III

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