Bart De Strooper

The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.

The amyloid cascade hypothesis, which posits that the deposition of the amyloid-β peptide in the brain is a central event in Alzheimers disease pathology, has dominated research for the past twenty years. Several therapeutics that were purported to reduce amyloid-β production or aggregation have failed in Phase III clinical testing, and many others are in various stages of development. Therefore, it is timely to review the science underpinning the amyloid cascade hypothesis, consider what type of clinical trials will constitute a valid test of this hypothesis and explore whether amyloid-β-directed therapeutics will provide the medicines that are urgently needed by society for treating this devastating disease.

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimers disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its β-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Lipids revert inert Aβ amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Aβ‐amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimers disease (AD), the role of mature Aβ‐fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds ‘forward’ in a near‐irreversible manner from the monomeric Aβ peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Aβ amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Aβ but rather toward soluble amyloid protofibrils. We characterized these ‘backward’ Aβ protofibrils generated from mature Aβ fibers and compared them with previously identified ‘forward’ Aβ protofibrils obtained from the aggregation of fresh Aβ monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Aβ‐aggregates that could readily be activated by exposure to biological lipids.

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and γ-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that β subunits of voltage-gated sodium channels are sequentially processed by BACE1 and γ-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture.

The γ‐secretase cleavage is the last step in the generation of the β‐amyloid peptide (Aβ) from the amyloid precursor protein (APP). The Aβ precipitates in the amyloid plaques in the brain of Alzheimers disease patients. The fate of the intracellular APP carboxy‐terminal stub generated together with Aβ has been, in contrast, only poorly documented. The analogies between the processing of APP and other transmembrane proteins like SREBP and Notch suggests that this intracellular fragment could have important signalling functions. We demonstrate here that APP‐C59 is rapidly degraded (half‐life ∼5 min) when overexpressed in baby hamster kidney cells or primary cultures of neurones by a mechanism that is not inhibited by endosomal/lysosomal or proteasome inhibitors. Furthermore, APP‐C59 binds to the DNA binding protein Fe65, although this does not increase the half‐life of APP‐C59. Finally, we demonstrate that a fraction of APP‐C59 becomes redistributed to the nuclear detergent‐insoluble pellet, in which the transcription factor SP1 is also present. Overall our results reinforce the analogy between Notch and APP processing, and suggest that the APP intracellular domain, like the Notch intracellular domain, could have a role in signalling events from the plasma membrane to the nucleus.

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

The role of G protein-coupled receptors in the pathology of Alzheimer's disease

G protein-coupled receptors (GPCRs) are involved in numerous key neurotransmitter systems in the brain that are disrupted in Alzheimers disease (AD). GPCRs also directly influence the amyloid cascade through modulation of the α-, β- and γ-secretases, proteolysis of the amyloid precursor protein (APP), and regulation of amyloid-β degradation. Additionally, amyloid-β has been shown to perturb GPCR function. Emerging insights into the mechanistic link between GPCRs and AD highlight the potential of this class of receptors as a therapeutic target for AD.

Presenilin-1 deficiency leads to loss of Cajal–Retzius neurons and cortical dysplasia similar to human type 2 lissencephaly

BACKGROUND Presenilin-1 (PS1) is a transmembrane protein that is located in the endoplasmic reticulum and the cis Golgi apparatus. Missense mutations of PS1 that modify gamma-secretase function, leading to a pathologic processing of amyloid precursor protein, are an important cause of familial Alzheimers disease. Physiologically, the presenilins are involved in the Notch and Wnt-beta-catenin signaling pathways. RESULTS PS1-deficient mice develop a cortical dysplasia resembling human type 2 lissencephaly, with leptomeningeal fibrosis and migration of cortical-plate neurons beyond their normal position into the marginal zone and subarachnoid space. This disorder of neuronal migration is associated with the disappearance of the majority of the cells of the marginal zone, notably most of the Cajal-Retzius pioneer neurons, between embryonic days E14 and E18, and is preceded and accompanied by disorganization of Notch-1 immunoreactivity on the neuronal cell membranes. The marginal zone also becomes depleted of the extracellular matrix protein reelin and chondroitin sulfate proteoglycans. At that stage PS1 is transiently expressed in leptomeningeal fibroblasts, which are mandatory for the trophic support of Cajal-Retzius neurons. CONCLUSIONS In agreement with models in which neuronal migration disorders have been linked to a defect in Cajal-Retzius cells, the loss of most of these cells in PS1-deficient mice leads to cortical dysplasia. Because PS1 is normally expressed in the leptomeninges, and these become fibrotic in the PS1-knockout mice, we favor the hypothesis that the loss of Cajal-Retzius cells is caused by a defective trophic interaction with leptomeningeal cells, possibly involving disruption of Notch signaling.

The Orphan G Protein–Coupled Receptor 3 Modulates Amyloid-Beta Peptide Generation in Neurons

Deposition of the amyloid-β peptide is a pathological hallmark of Alzheimers disease. A high-throughput functional genomics screen identified G protein–coupled receptor 3 (GPR3), a constitutively active orphan G protein–coupled receptor, as a modulator of amyloid-β production. Overexpression of GPR3 stimulated amyloid-β production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-β peptide in vitro and in an Alzheimers disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature γ-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimers disease and is elevated in the sporadic Alzheimers disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimers disease.

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.

Urea-based β-amyloid (Aβ) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Aβ peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Aβ peptides, namely, 1–40/42 and carboxyl-terminal-truncated 1–37, 1–38, and 1–39, was found. Besides Aβ1–42, we also observed a consistent elevation of amino-terminal-truncated Aβ2–42 in a detergent-soluble pool in brains of subjects with Alzheimers disease. Aβ2–42 was also specifically elevated in cerebrospinal fluid samples of Alzheimers disease patients. To decipher the contribution of potential different γ-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Aβ peptides, we overexpressed β-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1−/− neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1−/− neurons and PS1+/+ neurons overexpressing APP-Δct (a slow-internalizing mutant) show a decrease of all secreted Aβ peptide species, as expected, because this mutant is processed mainly by α-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Aβ2–42 is significantly less affected in PS1−/− neurons and in neurons transfected with the endocytosis-deficient APP-Δct construct. Our data confirm that PS1 is closely involved in the production of Aβ1–40/42 and the carboxyl-terminal-truncated Aβ1–37, Aβ1–38, and Aβ1–39, but the amino-terminal-truncated and carboxyl-terminal-elongated Aβ2–42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Aβ peptide species could be generated by a βAsp/Ala-secretase activity.