Chiaki Tanabe

Aβ43 Is the Earliest-Depositing Aβ Species in APP Transgenic Mouse Brain and Is Converted to Aβ41 by Two Active Domains of ACE

Amyloid-β protein (Aβ) varies in length at its carboxyl terminus. The longer Aβ species, Aβ43 and Aβ42, are highly amyloidogenic and deposit more frequently than Aβ40 in the brain of Alzheimer disease (AD) patients. However, the characterization of Aβ43 deposition in the brain and the relationship between Aβ43 and Aβ42 or Aβ40 remain unclear. We provide evidence that Aβ43 deposition appears earlier than Aβ42 and Aβ40 deposition in the brain of mutant amyloid precursor protein transgenic (APPtg) mice, suggesting that Aβ43 is the earliest-depositing species. In addition, we found increased Aβ43 levels and Aβ43/Aβ42 ratios in the serum of AD patients, suggesting their use as diagnostic blood biomarkers for AD. We further show that angiotensin-converting enzyme (ACE) converts Aβ43 to Aβ41. Notably, this Aβ43-to-Aβ41 converting activity requires two active domains of ACE. Inhibition of ACE activity significantly enhanced Aβ43 deposition in APPtg mouse brain. Our results suggest that Aβ43 is the earliest-depositing species in brain parenchyma and that Aβ43 may trigger later Aβ42 and Aβ40 deposition or may be converted to Aβ42 and Aβ40 plaques. Activities of both ACE domains may be important for reducing Aβ43 levels in serum and reducing brain Aβ43 deposition.

Conversion of Aβ43 to Aβ40 by the successive action of angiotensin-converting enzyme 2 and angiotensin-converting enzyme

The longer and neurotoxic species of amyloid‐β protein (Aβ), Aβ42 and Aβ43, contribute to Aβ accumulation in Alzheimers disease (AD) pathogenesis and are considered to be the primary cause of the disease. In contrast, the predominant secreted form of Aβ, Aβ40, inhibits amyloid deposition and may have neuroprotective effects. We have reported that angiotensin‐converting enzyme (ACE) converts Aβ42 to Aβ40 and that Aβ43 is the earliest‐depositing Aβ species in the amyloid precursor protein transgenic mouse brain. Here we found that Aβ43 can be converted to Aβ42 and to Aβ40 in mouse brain lysate. We further identified the brain Aβ43‐to‐Aβ42‐converting enzyme as ACE2. The purified human ACE2 converted Aβ43 to Aβ42, and this activity was inhibited by a specific ACE2 inhibitor, DX600. Notably, the combination of ACE2 and ACE could convert Aβ43 to Aβ40. Our results indicate that the longer, neurotoxic forms of Aβ can be converted to the shorter, less toxic or neuroprotective forms of Aβ by ACE2 and ACE. Moreover, we found that ACE2 activity showed a tendency to decrease in the serum of AD patients compared with normal controls, suggesting an association between lower ACE2 activity and AD. Thus, maintaining brain ACE2 and ACE activities may be important for preventing brain amyloid neurotoxicity and deposition in Alzheimers disease.

An E3 ubiquitin ligase, Synoviolin, is involved in the degradation of immature nicastrin, and regulates the production of amyloid β‐protein

The presenilin complex, consisting of presenilin, nicastrin, anterior pharynx defective‐1 and presenilin enhancer‐2, constitutes γ‐secretase, which is required for the generation of amyloid β‐protein. In this article, we show that Synoviolin (also called Hrd1), which is an E3 ubiquitin ligase implicated in endoplasmic reticulum‐associated degradation, is involved in the degradation of endogenous immature nicastrin, and affects amyloid β‐protein generation. It was found that the level of immature nicastrin was dramatically increased in synoviolin‐null cells as a result of the inhibition of degradation, but the accumulation of endogenous presenilin, anterior pharynx defective‐1 and presenilin enhancer‐2 was not changed. This was abolished by the transfection of exogenous Synoviolin. Moreover, nicastrin was co‐immunoprecipitated with Synoviolin, strongly suggesting that nicastrin is the substrate of Synoviolin. Interestingly, amyloid β‐protein generation was increased by the overexpression of Synoviolin, although the nicastrin level was decreased. Thus, Synoviolin‐mediated ubiquitination is involved in the degradation of immature nicastrin, and probably regulates amyloid β‐protein generation.

The Ubiquitin Ligase Synoviolin Up-regulates Amyloid β Production by Targeting a Negative Regulator of γ-Secretase, Rer1, for Degradation

Background: Synoviolin, a ubiquitin ligase, modulates the generation of Aβ produced by γ-secretase. Results: Rer1, a negative regulator of γ-secretase activity, is ubiquitinated and degraded by synoviolin. Conclusion: Synoviolin modulates the generation of Aβ by inducing the degradation of Rer1, thereby regulating assembly of the γ-secretase complex. Significance: Rer1 is a key factor in modulating the generation of Aβ by synoviolin, which is associated with ERAD. Alzheimers disease is characterized by the deposition of Aβ, which is generated from the amyloid precursor protein through its cleavage by β- and γ-secretases. The γ-secretase complex component nicastrin (NCT) plays significant roles in the assembly and proper trafficking of the γ-secretase complex and in the recognition of amyloid precursor protein. NCT is incorporated into the γ-secretase complex in the endoplasmic reticulum (ER) and glycosylated in the Golgi. In contrast, unassembled NCT is retrieved or retained in the ER by the protein Retention in endoplasmic reticulum 1 (Rer1). We reported previously that synoviolin (Syvn), an E3 ubiquitin ligase, degrades NCT and affects the generation of Aβ. Here, we examined in more detail the effect of Syvn on the generation of Aβ. We found that overexpression of a dominant negative form of Syvn (C307A mutant) and a Syvn-RNAi decreased the generation of Aβ. These results indicate that the ubiquitin ligase activity of Syvn up-regulates the generation of Aβ. We hypothesized, therefore, that Syvn regulates the assembly or localization of the γ-secretase complex by ubiquitinating Rer1, resulting in its subsequent degradation. Our findings that the level of Rer1 was increased in Syvn knockout fibroblasts because of inhibition of its degradation support this hypothesis. Moreover, we found that Rer1 interacts with Syvn in the ER, is ubiquitinated by Syvn, and is then degraded via the proteasome or lysosomal pathways. Finally, we showed that localization of mature NCT to the plasma membrane as well as γ-secretase complex levels are decreased in fibroblasts of Syvn knockout mice. Thus, it is likely that Syvn regulates the assembly of the γ-secretase complex via the degradation of Rer1, which results in the generation of Aβ.

Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology

Alzheimer’s disease is characterized by neuronal loss and cerebral accumulation of amyloid-β protein (Aβ) and lowering the generation of Aβ is a pivotal approach in the strategy of Alzheimer’s disease treatment. Midlife hypertension is a major risk factor for the future onset of sporadic Alzheimer’s disease and the use of some antihypertensive drugs may decrease the incidence of Alzheimer’s disease. However, it is largely unknown how the blood pressure regulation system is associated with the pathogenesis of Alzheimer’s disease. Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer’s disease. The lack of AT1a inhibited the endocleavage of presenilin-1 (PS1), which is essential for γ-secretase complex formation and Aβ generation. Notably, the ligand of AT1a, angiotensin II, enhanced Aβ generation, PS1 endocleavage and γ-secretase complex formation. Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation. Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer’s disease.

Inhibition by KMI-574 leads to dislocalization of BACE1 from lipid rafts

BACE1 initiates processing of the amyloid precursor protein (APP) in the production of amyloid β (Aβ) peptide. After β‐cleavage by BACE1, the C‐terminal stub of the APP fragment is further processed by the γ‐secretase complex to produce Aβ. Because APP, Aβ, the γ‐secretase complex, and BACE1 are found in lipid raft membranes, Aβ production is widely accepted to occur in lipid rafts. However, whether BACE1 is activated within the rafts is unclear. To analyze the relationship between the activity and the localization of BACE1, we used a new BACE1 inhibitor, KMI‐574, and separated raft membranes on sucrose density gradients. In the presence of KMI‐574, the localization of BACE1 shifted from the rafts to nonraft membranes in HEK293 cells. We also analyzed the proteolytically inactive mutants, D93A, D289A, and D93A/D289A, of BACE1. These mutants also moved from rafts to nonrafts, and the D93A/D289A double‐mutant localized exclusively to nonraft membranes. The mutants were defective in maturation by glynosylation and formed hyperoligomers, suggesting that the BACE1 oligomers could not exit from the ER and be transported to the Golgi apparatus. Our findings suggest that the activated conformation of BACE1 is important for protein transport and localization to lipid rafts.

Localization of mature neprilysin in lipid rafts

Alzheimers disease (AD) is characterized by senile plaques caused by amyloid‐β peptide (Aβ) accumulation. It has been reported that Aβ generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aβ‐degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aβ accumulates but does not modulate the protease activity of NEP.

ER-stress-inducible Herp, facilitates the degradation of immature nicastrin.

BACKGROUND Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein harboring an ubiquitin-like domain (ULD). However, its biological functions are not fully understood. Here, we examined the role of Herp in the degradation of γ-secretase components. METHODS Effects of ULD-lacking Herp (ΔUb-Herp) expression on the degradation of γ-secretase components were analyzed. RESULTS The cellular expression of ΔUb-Herp was found to inhibit the degradation of overexpressed immature nicastrin and full-length presenilin. The mechanisms underlying Herp-mediated nicastrin degradation was further analyzed. We found that immature nicastrin accumulates in the ER of ΔUb-Herp overexpressing cells or Herp-deficient cells more than that in the ER of wild-type cells. Further, ΔUb-Herp expression inhibited nicastrin ubiquitination, suggesting that the ULD of Herp is likely involved in nicastrin ubiquitination. Co-immunoprecipitation study showed that Herp as well as ΔUb-Herp potentially interacts with nicastrin, mediating nicastrin interaction with p97, which functions in retranslocation of misfolded proteins from the ER to the cytosol. CONCLUSIONS Thus, Herp is likely involved in degradation of immature nicastrin by facilitating p97-dependent nicastrin retranslocation and ubiquitination. GENERAL SIGNIFICANCE We suggest that Herp could play a role in the elimination of the excess unassembled components of a multimeric complex.

Differential effects of angiotensin II receptor blockers on Aβ generation.

Angiotensin II receptor blockers (ARBs) are widely prescribed for the medication of systemic hypertension and congestive heart failure. It has been reported that ARBs can reduce the risk for the onset of Alzheimers disease (AD) and have beneficial effects on dementia. Neurotoxic amyloid β-protein (Aβ) is believed to play a causative role in the development of AD. However, whether ARBs regulate Aβ generation remains largely unknown. Here, we studied the effect of ARBs on Aβ generation and found that telmisartan significantly increased Aβ40 and Aβ42 generation, but decreased the Aβ42/Aβ40 ratio. However, losartan, valsartan and candesartan did not increase Aβ generation, while olmesartan significantly increased Aβ42 generation. We also found that telmisartan increased the Aβ generation through angiotensin type 1a receptor (AT1a) and the receptor-related phosphotidylinositide 3-kinases (PI3K) pathway. Our findings revealed the different effects of ARBs on Aβ generation and provide new evidence for the relationship between antihypertensive treatment and AD pathogenesis.

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.