Recent epidemiological studies show a strong reduction in the incidence of Alzheimer's disease in patients treated with cholesterol-lowering statins. Moreover, elevated A␤42 levels and the 4 allele of the lipid-carrier apolipoprotein E are regarded as risk factors for sporadic and familial Alzheimer's disease. Here we demonstrate that the widely used cholesterol-lowering drugs simvastatin and lovastatin reduce intracellular and extracellular levels of A␤42 and A␤40 peptides in primary cultures of hippocampal neurons and mixed cortical neurons. Likewise, guinea pigs treated with high doses of simvastatin showed a strong and reversible reduction of cerebral A␤42 and A␤40 levels in the cerebrospinal fluid and brain homogenate. These results suggest that lipids are playing an important role in the development of Alzheimer's disease. Lowered levels of A␤42 may provide the mechanism for the observed reduced incidence of dementia in statin-treated patients and may open up avenues for therapeutic interventions.A part from age, environmental factors have only slight influence on the incidence of Alzheimer's disease (AD). Very recently, two independent reports showed a strong decrease in the incidence of AD and dementia for patients that were treated with statins (1, 2). Both studies were retrospective, and statins were not given in any relation to dementia. Usually statins are prescribed for treatment of elevated serum cholesterol levels in patients. They reduce cholesterol levels by inhibiting the bottleneck enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. They are widely used drugs, well characterized and considered to be very safe for long-time treatment, and approved for use in elderly patients (3, 4).The 4 allele of the apolipoprotein E (apoE) is the major genetic risk factor for AD (5). Several lines of evidence indicate that apoE 4 and statins have a related influence on AD. The normal cellular function of apoE is uptake and delivery of lipids. The isoform apoE 4 correlates with an increased risk for atherosclerosis (6) and amyloid plaque formation (7). Moreover, elevated cholesterol uptake increases amyloid plaque formation or amyloid deposition (8,9). This correlation may be extended to A␤ production, since cellular cholesterol levels affect neuronal A␤ production in vitro (10). Initially, A␤ has been a focus of AD research, because it was found to be the major constituent of the amyloid plaque. It is unknown whether the amyloid plaque is actively involved in the neurodegenerative process in AD or instead is a consequence of the disease process. More recently, however, A␤ has been a focus of AD research not because of it presence in the amyloid plaque, but because an overproduction of a minor A␤ isoform, A␤42, is linked to all identified inherited forms of AD (11-13).A␤ is produced during normal cellular processing of the Alzheimer amyloid precursor protein (APP) (14) by ␤-secretase and ␥-secretase (15). While the majority of all A␤ isoforms produced is A␤40, Ϸ10% of total A␤ ...

␣-synucleinopathy ͉ mass spectrometry ͉ proteolysis ͉ Lewy body P arkinson's disease (PD) is a common progressive neurodegenerative disease characterized by the loss of dopaminergic neurons of substantia nigra and the presence of the fibrillar cytoplasmic aggregates of ␣-synuclein (␣-Syn) in multiple brain regions (1, 2). Mutations in the ␣-Syn gene (3-7) and the abnormal aggregation of ␣-Syn are implicated in the pathogenesis of PD, and other related diseases are classified as ␣-synucleinopathies (1, 8-10). ␣-Syn is a highly conserved protein of 140 amino acids that is predominantly expressed in neurons, particularly in presynaptic terminals (11), and may have a role in synaptic plasticity and modulation of dopaminergic neurotransmission (11).Although the bulk of previous studies focused on the aggregation and the biology of the full-length ␣-Syn (␣-SynFL) (12, 13), the conspicuous presence of lower molecular mass ␣-Syn species in ␣-Syn aggregates (14, 15), and the enhanced in vitro fibril assembly of recombinant C-terminally truncated ␣-Syn (16, 17) suggests that the low-molecular mass ␣-Syn species may be of pathogenic significance. However, because postpathogenic and͞or postmortem processes could potentially generate a variety of ␣-Syn species, the significance of low-molecular mass ␣-Syn species to the development of ␣-synucleinopathy is uncertain.Herein, we demonstrate that C-terminally truncated lowmolecular mass ␣-Syn species (␣-Syn⌬C) with aggregationpromoting properties are normally generated in vivo. The expression of familial PD (FPD)-linked mutant human (Hu) ␣-Syn is associated with the higher cellular accumulation of ␣-Syn⌬C. Moreover, human cases with ␣-Syn lesions show preferential accumulation of ␣-Syn⌬C in aggregates and higher relative levels of soluble ␣-Syn⌬C. Our findings show that ␣-Syn⌬Cs are not an artifact of postpathologic processes and are likely to participate in the disease-linked aggregation of ␣-Syn. Materials and MethodsAdditional details are provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

The betaA4 peptide, a major component of senile plaques in Alzheimer's disease (AD) brain, has been found in cerebrospinal fluid (CSF) and blood of both AD patients and normal subjects. Although betaA4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed betaA4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of betaA4 1-40 and 1-42, and used them for the specific Western blot detection. After optimizing the assay conditions, these antibodies detected low picogram amount of betaA4, and both betaA4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. Blood levels of betaA4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF betaA4 1-42 level is lower in AD patients compared with non-demented controls, although there was a significant overlap between the groups. The level of betaA4 1-40 in CSF, and of betaA4 1-40 as well as betaA4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length betaA4 band, we could also detect several N-terminal variants of betaA4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated betaA4 species migrating at the position of 3.3 and 3.7 kDa were found in CSF, while 3.7- and 5-kDa forms were found in plasma. The relative abundance of these various species were considerably different in the CSF and plasma, suggesting that the cellular source and/or clearance of betaA4 is different in these two compartments.

Amyloid beta peptide (Abeta) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Abeta and of the amyloid precursor protein (APP) is unknown. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Abeta42 directly activates neutral SMase and downregulates SM levels, whereas Abeta40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered Abeta40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).

In a randomized, placebo-controlled, double-blind study, we investigated whether statins alter cholesterol metabolites and reduce Abeta levels in the cerebrospinal fluid of 44 patients with Alzheimer's disease. Individuals were given up to 80mg simvastatin daily or placebo for 26 weeks. Overall, simvastatin did not significantly alter cerebrospinal fluid levels of Abeta40 and Abeta42. In post hoc analysis, simvastatin significantly decreased Abeta40 levels in the cerebrospinal fluid of patients with mild Alzheimer's disease. The reduction of Abeta40 correlated with the reduction of 24S-hydroxycholesterol. These changes were not observed in more severely affected patients.

Microglia activated by extracellularly deposited amyloid β peptide (Aβ) act as a two-edged sword in Alzheimer’s disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing Aβ via phagocytosis. TLRs are associated with Aβ-induced microglial inflammatory activation and Aβ internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human Aβ (Aβ42). TLR2 deficiency reduced Aβ42-triggered inflammatory activation but enhanced Aβ phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to Aβ42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741–744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated Aβ42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimer’s amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for Aβ to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimer’s disease pathogenesis.

SummaryBackgroundNutrition is an important modifiable risk factor in Alzheimer's disease. Previous trials of the multinutrient Fortasyn Connect showed benefits in mild Alzheimer's disease dementia. LipiDiDiet investigated the effects of Fortasyn Connect on cognition and related measures in prodromal Alzheimer's disease. Here, we report the 24-month results of the trial.MethodsLipiDiDiet was a 24-month randomised, controlled, double-blind, parallel-group, multicentre trial (11 sites in Finland, Germany, the Netherlands, and Sweden), with optional 12-month double-blind extensions. The trial enrolled individuals with prodromal Alzheimer's disease, defined according to the International Working Group (IWG)-1 criteria. Participants were randomly assigned (1:1) to active product (125 mL once-a-day drink containing Fortasyn Connect) or control product. Randomisation was computer-generated centrally in blocks of four, stratified by site. All study personnel and participants were masked to treatment assignment. The primary endpoint was change in a neuropsychological test battery (NTB) score. Analysis was by modified intention to treat. Safety analyses included all participants who consumed at least one study product dose. This trial is registered with the Dutch Trial Register, number NTR1705.FindingsBetween April 20, 2009, and July 3, 2013, 311 of 382 participants screened were randomly assigned to the active group (n=153) or control group (n=158). Mean change in NTB primary endpoint was −0·028 (SD 0·453) in the active group and −0·108 (0·528) in the control group; estimated mean treatment difference was 0·098 (95% CI −0·041 to 0·237; p=0·166). The decline in the control group was less than the prestudy estimate of −0·4 during 24 months. 66 (21%) participants dropped out of the study. Serious adverse events occurred in 34 (22%) participants in the active group and 30 (19%) in control group (p=0·487), none of which were regarded as related to the study intervention.InterpretationThe intervention had no significant effect on the NTB primary endpoint over 2 years in prodromal Alzheimer's disease. However, cognitive decline in this population was much lower than expected, rendering the primary endpoint inadequately powered. Group differences on secondary endpoints of disease progression measuring cognition and function and hippocampal atrophy were observed. Further study of nutritional approaches with larger sample sizes, longer duration, or a primary endpoint more sensitive in this pre-dementia population, is needed.FundingEuropean Commission 7th Framework Programme.