Yukiko Hori

BACE1 Activity Is Modulated by Cell-Associated Sphingosine-1-Phosphate

Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to generate sphingosine-1-phosphate (S1P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of β-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for amyloid-β peptide (Aβ) production, is modulated by S1P in mouse neurons. Treatment by SphK inhibitor, RNA interference knockdown of SphK, or overexpression of S1P degrading enzymes decreased BACE1 activity, which reduced Aβ production. S1P specifically bound to full-length BACE1 and increased its proteolytic activity, suggesting that cellular S1P directly modulates BACE1 activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimers disease. The unique modulatory effect of cellular S1P on BACE1 activity is a novel potential therapeutic target for Alzheimers disease.

Aβ Immunotherapy: Intracerebral Sequestration of Aβ by an Anti-Aβ Monoclonal Antibody 266 with High Affinity to Soluble Aβ

Amyloid β (Aβ) immunotherapy is emerging as a promising disease-modifying therapy for Alzheimers disease, although the precise mechanisms whereby anti-Aβ antibodies act against amyloid deposition and cognitive deficits remain elusive. To test the “peripheral sink” theory, which postulates that the effects of anti-Aβ antibodies in the systemic circulation are to promote the Aβ efflux from brain to blood, we studied the clearance of 125I-Aβ1-40 microinjected into mouse brains after intraperitoneal administration of an anti-Aβ monoclonal antibody 266. 125I-Aβ1-40 was rapidly eliminated from brains with a half-life of ∼30 min in control mice, whereas 266 significantly retarded the elimination of Aβ, presumably due to formation of Aβ-antibody complex in brains. Administration of 266 to APP transgenic mice increased the levels of monomer Aβ species in an antibody-bound form, without affecting that of total Aβ. We propose a novel mechanism of Aβ immunotherapy by the class of anti-Aβ antibodies that preferentially bind soluble Aβ, i.e., intracerebral, rather than peripheral, sequestration of soluble, monomer form of Aβ, thereby preventing the accumulation of multimeric toxic Aβ species in brains.

The tottori (D7N) and English (H6R) familial Alzheimer disease mutations accelerate Aβ fibril formation without increasing protofibril formation

A subset of Alzheimer disease cases is caused by autosomal dominant mutations in genes encoding the amyloid β-protein precursor or presenilins. Whereas some amyloid β-protein precursor mutations alter its metabolism through effects on Aβ production, the pathogenic effects of those that alter amino acid residues within the Aβ sequence are not fully understood. Here we examined the biophysical effects of two recently described intra-Aβ mutations linked to early-onset familial Alzheimer disease, the D7N Tottori-Japanese and H6R English mutations. Although these mutations do not affect Aβ production, synthetic Aβ(1-42) peptides carrying D7N or H6R substitutions show enhanced fibril formation. In vitro analysis using Aβ(1-40)-based mutant peptides reveal that D7N or H6R mutations do not accelerate the nucleation phase but selectively promote the elongation phase of amyloid fibril formation. Notably, the levels of protofibrils generated from D7N or H6R Aβ were markedly inhibited despite enhanced fibril formation. These N-terminal Aβ mutations may accelerate amyloid fibril formation by a unique mechanism causing structural changes of Aβ peptides, specifically promoting the elongation process of amyloid fibrils without increasing metastable intermediates.

Microfluidic Chemotaxis Platform for Differentiating the Roles of Soluble and Bound Amyloid-β on Microglial Accumulation

Progressive microglial accumulation at amyloid-β (Aβ) plaques is a well-established signature of the pathology of Alzheimers disease, but how and why microglia accumulate in the vicinity of Aβ plaques is unknown. To understand the distinct roles of Aβ on microglial accumulation, we quantified microglial responses to week-long lasting gradients of soluble Aβ and patterns of surface-bound Aβ in microfluidic chemotaxis platforms. We found that human microglia chemotaxis in gradients of soluble Aβ42 was most effective at two distinct concentrations of 23 pg.mL−1 and 23 ng.mL−1 Aβ42 in monomers and oligomers. We uncovered that while the chemotaxis at higher Aβ concentrations was exclusively due to Aβ gradients, chemotaxis at lower concentrations was enhanced by Aβ-induced microglial production of MCP-1. Microglial migration was inhibited by surface-bound Aβ42 in oligomers and fibrils above 45 pg.mm−2. Better understanding of microglial migration can provide insights into the pathophysiology of senile plaques in AD.

A Food and Drug Administration-approved Asthma Therapeutic Agent Impacts Amyloid β in the Brain in a Transgenic Model of Alzheimer Disease

Background: Cromolyn sodium is an FDA-approved drug structurally similar to fisetin, an antiamyloidogenic molecule. Results: Cromolyn sodium interferes with amyloid β (Aβ) aggregation in vitro while rapidly decreasing the levels of soluble Aβ peptides in vivo after a week. Conclusion: Cromolyn sodium may have an impact on amyloid economy. Significance: Developing new disease-modifying therapeutics remains an urgent need in the treatment of Alzheimer disease. Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.

Role of apolipoprotein E in β-amyloidogenesis: isoform-specific effects on protofibril to fibril conversion of Aβ in vitro and brain Aβ deposition in vivo.

Background: ApoE is a genetic risk factor for Alzheimer disease. Results: As compared with apoE2/3, apoE4 failed to inhibit the conversion of Aβ protofibrils to fibrils in vitro. Intracerebral injection of Aβ protofibrils with apoE3 attenuated Aβ deposition, whereas apoE4 did not. Conclusion: ApoE3, not apoE4, impedes β-amyloid formation. Significance: Interaction between Aβ and apoE is a critical determinant of β-amyloid formation. Human APOE ϵ4 allele is a strong genetic risk factor of Alzheimer disease. Neuropathological and genetic studies suggested that apolipoprotein E4 (apoE4) protein facilitates deposition of amyloid β peptide (Aβ) in the brain, although the mechanism whereby apoE4 increases amyloid aggregates remains elusive. Here we show that injection of Aβ protofibrils induced Aβ deposition in the brain of APP transgenic mice, suggesting that Aβ protofibrils acted as a seed for aggregation and deposition of Aβ in vivo. Injection of Aβ protofibrils together with apoE3 significantly attenuated Aβ deposition, whereas apoE4 did not have this effect. In vitro assays revealed that the conversion of Aβ protofibrils to fibrils progressed more slowly upon coincubation with apoE2 or apoE3 compared with that with apoE4. Aβ protofibrils complexed with apoE4 were less stable than those with apoE2 or apoE3. These data suggest that the suppression effect of apoE2 or apoE3 on the structural conversion of Aβ protofibrils to fibrils is stronger than those of apoE4, thereby impeding β-amyloid deposition.

Partial loss of CALM function reduces Aβ42 production and amyloid deposition in vivo

Aberrant production, clearance and deposition of amyloid-β protein (Aβ) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). γ-Secretase is the enzyme responsible for generating various Aβ species, such as Aβ40 and toxic Aβ42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of Aβ42, through the regulation of the clathrin-mediated endocytosis of γ-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of γ-secretase and to the Aβ42 production ratio. Moreover, reduction of CALM decreases Aβ deposition as well as brain levels of insoluble Aβ42 in vivo These results suggest that CALM expression modifies AD risk by regulating Aβ pathology.

Loss of kallikrein‐related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice

Deposition of amyloid‐β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimers disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein‐related peptidase 7 (KLK7) as an astrocyte‐derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S‐positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatment in the primary astrocyte, suggesting that Klk7 is homeostatically modulated by Aβ‐induced responses. Finally, we found that the Food and Drug Administration‐approved anti‐dementia drug memantine can increase the expression of Klk7 and Aβ degradation activity specifically in the astrocytes. These data suggest that KLK7 is an important enzyme in the degradation and clearance of deposited Aβ species by astrocytes involved in the pathogenesis of AD.

Retrograde transport of γ-secretase from endosomes to the trans-Golgi network regulates Aβ42 production

The aberrant metabolism of amyloid‐β protein (Aβ) in the human brain has been implicated in the etiology of Alzheimer disease (AD). γ‐Secretase is the enzyme that generates various forms of Aβ, such as Aβ40 and Aβ42, the latter being an aggregation‐prone toxic peptide that is involved in the pathogenesis of AD. Recently, we found that clathrin‐mediated endocytosis of γ‐secretase affects the production and deposition of Aβ42 in vivo, suggesting that the membrane trafficking of γ‐secretase affects its enzymatic activity. However, the detailed intracellular trafficking pathway of γ‐secretase and its contribution to Aβ42 generation remain unclear. Here, we show that Retro‐2, which inhibits the retrograde transport, elevated the Aβ42‐generating activity both in cultured cells and mice brain. However, the result of in vitro γ‐secretase assay using a recombinant substrate suggested that Retro‐2 did not elevate the intrinsic Aβ42‐production activity of γ‐secretase. Immunocytochemistry and cell‐surface biotinylation experiments revealed that γ‐secretase is recycled via the endosome‐to‐trans‐Golgi network transport. In addition, γ‐secretase is retrogradely transported by syntaxin 5/6, known as targets of Retro‐2, independent pathway. Conversely, TPT‐260, which enhances the trafficking function of retromers, lowered Aβ42 levels and the Aβ42/(Aβ40 + Aβ42) ratio in secreted Aβ from cultured cells. Our results strongly suggest that the endosome‐to‐trans‐Golgi network trafficking of γ‐secretase regulates its Aβ42 production activity. Modulation of this trafficking pathway might be a potential target for the development of Aβ42‐lowering AD therapeutics.

GENOMEWIDE SCREENING OF MOLECULES INVOLVED IN UPTAKE OF ALZHEIMER DISEASE-ASSOCIATED PROTEINS BY CRIPSR/CAS9 SYSTEM

Background: Fifty percent of amyloid-b (Ab) in the human central nervous system is cleared via direct transport across the blood-brain barrier (BBB) and reabsorption from CSF into the blood. This suggests thatmodulatingAb clearance from the blood could be a possible new therapeutic strategy for AD. Resident immune cells in the brain can participate in Ab clearance via phagocytosis and proteolytic degradation, but the role of peripheral immune cells in Ab clearance from blood remains unknown. Therefore the aim of this study was to determine phagocytic capacity of Ab and the expression of two important Ab degrading enzymes, neprilysin and insulin-degrading enzyme (IDE), in white blood cells.Methods:For this proof of principle, total white blood cells were isolated fromwhole blood of anonymous donors visiting the VU University Medical center in Amsterdam, The Netherlands. A method to measure Ab40 and Ab42 phagocytosis was developed. Total white blood cells were exposed to fluorescent Ab for 30 minutes and subsequently washed. Next, extracellular Ab was quenched with 0.2% trypan blue and Ab phagocytosis was detected with flow cytometry. Neprilysin and IDE activity was measured in lysates of monocytes, lymphocytes and granulocytes with in-house activity assays. Results:Here we report the measurement of specific Ab clearance capacities in peripheral immune cells. Flow cytometry showed that both granulocytes and monocytes possess Ab40 and Ab42 phagocytic activity. Interestingly, neprilysin activity was highest in granulocytes followed by monocytes and lymphocytes (2.8, 0.04 and 0.01 mMneprilysin activity per 10.000 cells respectively). IDE activity was also highest in granulocytes followed by lymphocytes, but absent in monocytes (0.1 ng IDE and 0.02 ng IDE per 10.000 cells respectively). This shows that monocytes, lymphocytes and granulocytes all participate in peripheral Ab clearance. Conclusions:Next to monocytes and lymphocytes, we identified granulocytes as peripheral immune cells that possess high capacity to degrade Ab and to phagocytose both Ab40 and Ab42. As Granulocytes outnumber monocytes and lymphocytes in blood, they could be key players in Ab clearance.