Suppression of amyloid-ß secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-ß (Aß) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aß in mouse neurons. CLA treatment did not affect the level of ß-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aß protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aß generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aß in neurons.

A clinical dose of angiotensin-converting enzyme (ACE) inhibitor and heterozygous ACE deletion exacerbate Alzheimer's disease pathology in mice.

Inhibition of angiotensin-converting enzyme (ACE) is a strategy used worldwide for managing hypertension. In addition to converting angiotensin I to angiotensin II, ACE also converts neurotoxic ß-amyloid protein 42 (Aß42) to Aß40. Because of its neurotoxicity, Aß42 is believed to play a causative role in the development of Alzheimer's disease (AD), whereas Aß40 has neuroprotective effects against Aß42 aggregation and also against metal-induced oxidative damage. Whether ACE inhibition enhances Aß42 aggregation or impairs human cognitive ability are very important issues for preventing AD onset and for optimal hypertension management. In an 8-year longitudinal study, we found here that the mean intelligence quotient of male, but not female, hypertensive patients taking ACE inhibitors declined more rapidly than that of others taking no ACE inhibitors. Moreover, the sera of all AD patients exhibited a decrease in Aß42-to-Aß40-converting activity compared with sera from age-matched healthy individuals. Using human amyloid precursor protein transgenic mice, we found that a clinical dose of an ACE inhibitor was sufficient to increase brain amyloid deposition. We also generated human amyloid precursor protein/ACE+/- mice and found that a decrease in ACE levels promoted Aß42 deposition and increased the number of apoptotic neurons. These results suggest that inhibition of ACE activity is a risk factor for impaired human cognition and for triggering AD onset.

Interaction between Angiotensin Receptor and ß-Adrenergic Receptor Regulates the Production of Amyloid ß-Protein.

Alzheimer's disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid ß-protein (Aß) within the parenchyma of the brain. Aß is considered to be the key pathogenic factor of AD. Recently, we showed that Angiotensin II type 1 receptor (AT1R), which regulates blood pressure, is involved in Aß production, and that telmisartan (Telm), which is an angiotensin II receptor blocker (ARB), increased Aß production via AT1R. However, the precise mechanism underlying how AT1R is involved in Aß production is unknown. Interestingly, AT1R, a G protein-coupled receptor, was strongly suggested to be involved in signal transduction by heterodimerization with ß2-adrenergic receptor (ß2-AR), which is also shown to be involved in Aß generation. Therefore, in this study, we aimed to clarify whether the interaction between AT1R and ß2-AR is involved in the regulation of Aß production. To address this, we analyzed whether the increase in Aß production by Telm treatment is affected by ß-AR antagonist using fibroblasts overexpressing amyloid precursor protein (APP). We found that the increase in Aß production by Telm treatment was decreased by the treatment of ß2-AR selective antagonist ICI-118551 more strongly than the treatment of ß1-AR selective antagonists. Furthermore, deficiency of AT1R abolished the effect of ß2-AR antagonist on the stimulation of Aß production caused by Telm. Taken together, the interaction between AT1R and ß2-AR is likely to be involved in Aß production.

Iron treatment inhibits Aß42 deposition in vivo and reduces Aß42/Aß40 ratio.

Alzheimer's disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid ß-protein (Aß) within the parenchyma of the brain. Aß42, which is 42 amino acids in length, is considered to be the key pathogenic factor in AD. Iron deposition is found abundantly in the amyloid plaques of AD patients; however, whether iron intake exacerbates amyloid deposition in vivo is unknown. Here, we treated AD model mice with iron-containing water and found that Aß42 deposition in the brain was significantly inhibited, along with a decrease in iron deposition. Iron treatment did not change the overall levels of iron in the brain or serum. Interestingly, Aß40 generation was significantly increased by iron treatment in amyloid precursor protein (APP)-overexpressing fibroblasts, whereas Aß42 generation did not change, which led to a decreased Aß42/Aß40 ratio. Because Aß40 can inhibit Aß42 aggregation in vitro, and Aß40 inhibits amyloid formation in vivo, our results suggest that iron can selectively enhances Aß40 generation and inhibit amyloid deposition by reducing the Aß42/Aß40 ratio. Thus, iron may be used as a novel treatment for reducing the Aß42/Aß40 ratio and Aß42 deposition in AD.

An E3 Ubiquitin Ligase, Synoviolin, Is Involved in the Degradation of Homocysteine-Inducible Endoplasmic Reticulum Protein.

Homocysteine-inducible endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible membrane protein involved in ER-associated degradation. Herp expression is maintained at low levels through a strict regulatory mechanism, but the details of this mechanism and the reasons why Herp expression is restricted in this manner remain unclear. Here, we show that Herp degradation involves synoviolin, an ER-resident E3 ubiquitin ligase. Herp protein levels were found to be markedly elevated in synoviolin-null cells, and Herp expression decreased when synoviolin was overexpressed. However, the lysine residues of Herp, which are ubiquitinated by E3 ubiquitin ligase, were not sufficient for regulation of Herp degradation. These results suggest that Herp degradation is mediated via synoviolin and that Herp ubiquitination involves amino acids other than lysine.

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein.

Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin.

Conversion of Aß43 to Aß40 by the successive action of angiotensin-converting enzyme 2 and angiotensin-converting enzyme.

The longer and neurotoxic species of amyloid-ß protein (Aß), Aß42 and Aß43, contribute to Aß accumulation in Alzheimer's disease (AD) pathogenesis and are considered to be the primary cause of the disease. In contrast, the predominant secreted form of Aß, Aß40, inhibits amyloid deposition and may have neuroprotective effects. We have reported that angiotensin-converting enzyme (ACE) converts Aß42 to Aß40 and that Aß43 is the earliest-depositing Aß species in the amyloid precursor protein transgenic mouse brain. Here we found that Aß43 can be converted to Aß42 and to Aß40 in mouse brain lysate. We further identified the brain Aß43-to-Aß42-converting enzyme as ACE2. The purified human ACE2 converted Aß43 to Aß42, and this activity was inhibited by a specific ACE2 inhibitor, DX600. Notably, the combination of ACE2 and ACE could convert Aß43 to Aß40. Our results indicate that the longer, neurotoxic forms of Aß can be converted to the shorter, less toxic or neuroprotective forms of Aß 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 Alzheimer's disease.

Aß43 is the earliest-depositing Aß species in APP transgenic mouse brain and is converted to Aß41 by two active domains of ACE.

Amyloid-ß protein (Aß) varies in length at its carboxyl terminus. The longer Aß species, Aß43 and Aß42, are highly amyloidogenic and deposit more frequently than Aß40 in the brain of Alzheimer disease (AD) patients. However, the characterization of Aß43 deposition in the brain and the relationship between Aß43 and Aß42 or Aß40 remain unclear. We provide evidence that Aß43 deposition appears earlier than Aß42 and Aß40 deposition in the brain of mutant amyloid precursor protein transgenic (APPtg) mice, suggesting that Aß43 is the earliest-depositing species. In addition, we found increased Aß43 levels and Aß43/Aß42 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 Aß43 to Aß41. Notably, this Aß43-to-Aß41 converting activity requires two active domains of ACE. Inhibition of ACE activity significantly enhanced Aß43 deposition in APPtg mouse brain. Our results suggest that Aß43 is the earliest-depositing species in brain parenchyma and that Aß43 may trigger later Aß42 and Aß40 deposition or may be converted to Aß42 and Aß40 plaques. Activities of both ACE domains may be important for reducing Aß43 levels in serum and reducing brain Aß43 deposition.

The ubiquitin ligase synoviolin up-regulates amyloid ß production by targeting a negative regulator of γ-secretase, Rer1, for degradation.

Alzheimer's disease is characterized by the deposition of Aß, which is generated from the amyloid precursor protein through its cleavage by ß- 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 Aß. Here, we examined in more detail the effect of Syvn on the generation of Aß. We found that overexpression of a dominant negative form of Syvn (C307A mutant) and a Syvn-RNAi decreased the generation of Aß. These results indicate that the ubiquitin ligase activity of Syvn up-regulates the generation of Aß. 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 Aß.

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.

Presenilin Deficiency Increases Susceptibility to Oxidative Damage in Fibroblasts.

Alzheimer's disease (AD) is a genetic and sporadic neurodegenerative disease characterized by extracellular amyloid-ß-protein (Aß) aggregates as amyloid plaques and neuronal loss in the brain parenchyma of patients. Familial AD (FAD) is found to be genetically linked to missense mutations either in presenilin (PS) or amyloid precursor protein (APP). Most of PS mutations increase Aß42/Aß40 ratio, which is thought to result in early amyloid deposition in brain. However, PS deficiency in the fore brain of adult mouse leads to neuronal loss in an Aß independent manner and the underlying mechanism is largely unknown. In this study, we found that reactive oxygen species (ROS) are increased in PS deficient fibroblasts and that H2O2 and ferrous sulfate treatment produced more ROS in PS deficient fibroblasts than in wild-type fibroblasts. PS deficient fibroblasts showed significantly decreased cellular ferritin levels compared with wild-type fibroblasts, suggesting reduced iron sequestrating capability in PS deficient cells. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ferritin levels, indicating that γ-secretase activity is important for maintaining its levels. Moreover, overexpression PS1 mutants in wild-type fibroblasts decreased ferritin light chain levels and enhanced intracellular ROS levels. Our results suggest that dysfunction of PS may reduce intracellular ferritin levels and is involved in AD pathogenesis through increasing susceptibility to oxidative damage.

Homocysteine, another risk factor for Alzheimer disease, impairs apolipoprotein E3 function.

Apolipoprotein E (apoE) ε4 and hyperhomocysteinemia are risk factors for Alzheimer disease (AD). The dimerization of apoE3 by disulfide bonds between cysteine residues enhances apoE3 function to generate HDL. Because homocysteine (Hcy) harbors a thiol group, we examined whether Hcy interferes with the dimerization of apoE3 and thereby impairs apoE3 function. We found that Hcy inhibits the dimerization of apoE3 and reduces apoE3-mediated HDL generation to a level similar to that by apoE4, whereas Hcy does not affect apoE4 function. Western blot analysis of cerebrospinal fluid showed that the ratio of apoE3 dimers was significantly lower in the samples from the patients with hyperhomocysteinemia than in those that from control subjects. Hyperhomocysteinemia induced by subcutaneous injection of Hcy to apoE3 knock-in mice decreased the level of the apoE3 dimer in the brain homogenate. Because apoE-HDL plays a role in amyloid ß-protein clearance, these results suggest that two different risk factors, apoE4 and hyperhomocysteinemia, may share a common mechanism that accelerates the pathogenesis of AD in terms of reduced HDL generation.

Dietary cis-9, trans-11-conjugated linoleic acid reduces amyloid ß-protein accumulation and upregulates anti-inflammatory cytokines in an Alzheimer's disease mouse model.

Conjugated linoleic acid (CLA) is an isomer of linoleic acid (LA). The predominant dietary CLA is cis-9, trans-11-CLA (c-9, t-11-CLA), which constitutes up to ~ 90% of total CLA and is thought to be responsible for the positive health benefits associated with CLA. However, the effects of c-9, t-11-CLA on Alzheimer's disease (AD) remain to be elucidated. In this study, we investigated the effect of dietary intake of c-9, t-11-CLA on the pathogenesis of an AD mouse model. We found that c-9, t-11-CLA diet-fed AD model mice significantly exhibited (1) a decrease in amyloid-ß protein (Aß) levels in the hippocampus, (2) an increase in the number of microglia, and (3) an increase in the number of astrocytes expressing the anti-inflammatory cytokines, interleukin-10 and 19 (IL-10, IL-19), with no change in the total number of astrocytes. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatographic analysis revealed that the levels of lysophosphatidylcholine (LPC) containing c-9, t-11-CLA (CLA-LPC) and free c-9, t-11-CLA were significantly increased in the brain of c-9, t-11-CLA diet-fed mice. Thus, dietary c-9, t-11-CLA entered the brain and appeared to exhibit beneficial effects on AD, including a decrease in Aß levels and suppression of inflammation.

Abeta42-to-Abeta40- and angiotensin-converting activities in different domains of angiotensin-converting enzyme.

Amyloid beta-protein 1-42 (Abeta42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Abeta. In contrast, Abeta40 is the predominant secreted form of Abeta and recent studies have suggested that Abeta40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Abeta42 to Abeta40, and its inhibition enhances brain Abeta42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628-8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Abeta42 to Abeta40. Interestingly, Abeta42-to-Abeta40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Abeta42-to-Abeta40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Abeta42-to-Abeta40-converting activity of ACE or increasing neurotoxic Abeta42.

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.

Engulfment of Toxic Amyloid ß-protein in Neurons and Astrocytes Mediated by MEGF10.

Amyloid-ß proteins (A ß), including Aß42 and A ß 43, are known pathogenesis factors of Alzheimer's disease (AD). Unwanted substances in the brain, including A ß, are generally removed by microglia, astrocytes, or neurons via a phagocytosis receptor. We observed that neurons and astrocytes engulfed A ß 42 and A ß 43, which are more neurotoxic than A ß 40. We previously showed that multiple-EGF like domains 10 (MEGF10) plays an important role in apoptotic cell elimination and is expressed in mammalian neurons and astrocytes. Therefore, we assessed whether MEGF10 is involved in A ß42 and A ß43 engulfment in MEGF10-expressing neurons and astrocytes. We found that MEGF10-expressing astrocytes and neurons engulfed A ß42 and A ß43 but not A ß40. Furthermore, incubation of the neurons and astrocytes with A ß42 and A ß43a ugmented MEGF10 phosphorylation; however, incubation with A ß40 did not have this augmenting effect. Our findings suggest that MEGF10 plays a phagocytosis receptor function for A ß42 and A ß43 in neurons and astrocytes.

A family of membrane proteins associated with presenilin expression and gamma-secretase function.

Presenilin 1 (PS1) forms the gamma-secretase complex with at least three components: nicastrin, APH-1, and PEN-2. This complex mediates intramembrane cleavage of amyloid precursor protein (APP) to generate beta-amyloid protein (Abeta) as well as other type 1 transmembrane proteins. Although PS1 mutations linked to familial Alzheimer's disease influence these cleavages, their biological consequences have not been fully understood. In this study, we used mRNA differential display analysis to identify a gene, denoted adoplin-1/ORMDL-1, which displays significantly reduced expression in association with PS1 mutations. Adoplin-1 and two highly homologous genes (adoplin-2, -3) constitute a gene family that encodes transmembrane proteins. The mRNA and protein levels of adoplins (particularly adoplin-1, -2) were markedly elevated in PS-deficient fibroblasts, compared to wild-type cells. Moreover, knockdown of the three adoplins by RNA interference affected maturation of nicastrin and its association with PS1. Adoplin knockdown additionally resulted in elevated levels of APP C-terminal fragments and decreased Abeta production, suggestive of reduced gamma-secretase activity. Our data collectively indicate that adoplins are unique molecules with PS-related expression and functions that may play important role(s) in the maturation and activity of the gamma-secretase complex.

Expression of MEGF10 in cholinergic and glutamatergic neurons.

Multiple-EGF like domains 10 (MEGF10) is the mammalian homologue of Draper, a Drosophila phagocytosis receptor that plays an important role in synapse elimination and cell type-specific recognition. However, the expression and function of MEGF10 in the brain remain to be elucidated. Therefore, we aimed to clarify the regions and types of neurons that express MEGF10 in the brain, and to determine whether cells expressing MEGF10 possess phagocytic abilities. Our results indicated that MEGF10 is expressed in cholinergic and glutamatergic neurons of the cortex, hippocampus, and substantia nigra. Furthermore, the ratio of neurons expressing MEGF10 was higher in the cortex and hippocampus of hAPP transgenic mice than in those of wild-type mice. Phagocytic activity was also observed in neuronal cells expressing MEGF10. Thus, our results indicate that MEGF10 may be responsible for phagocytic activity targeted toward unwanted substances such as amyloid in cholinergic and glutamatergic neurons.

Characterization of APH-1 mutants with a disrupted transmembrane GxxxG motif.

APH-1 is one of the four essential components of presenilin (PS)-gamma-secretase complexes. There are three major isoforms of APH-1 in humans: APH-1aS, APH-1aL, and APH-1b. To gain insight into the functional role of APH-1 in gamma-secretase complexes, we analyzed the relationship between the three APH-1 forms and characterized APH-1 mutants with a disrupted transmembrane GxxxG motif. We found that overexpression of APH-1aS or APH-1b in human cells significantly reduced the levels of endogenous APH-1aL protein. However, this displacement was not observed in PS-deficient cells, suggesting that it is dependent on PS. In transiently transfected cells, the levels of APH-1aL with G122D or L123D mutations were much lower than wild-type APH-1aL. Also, cycloheximide treatment of stable transfectants revealed that the mutant proteins are much less stable than the wild type. Furthermore, coimmunoprecipitation analysis showed that wild-type but not the mutant APH-1aL is incorporated into PS1 complexes, displacing endogenous APH-1aS. These results collectively indicate that the three forms of APH-1 can replace each other in PS complexes and that the transmembrane GxxxG region is essential for the stability of the APH-1 protein as well as the assembly of PS complexes.

ATP increases the migration of microglia across the brain endothelial cell monolayer.

The cerebral microcapillary endothelium, known as the blood-brain barrier (BBB), acts as a barrier between the blood and the interstitial fluid of the brain. The BBB therefore controls the passage of nutrients into the central nervous system (CNS). Microglia show a specific affinity for migration into the CNS, and this migration appears to occur independently of BBB integrity. To study the migration of microglia across the BBB, we developed an in vitro co-culture system of mouse brain endothelial cells (MBECs) and Ra2 microglia using Transwell inserts. We first investigated the influence of microglia or ATP, a microglial chemotactic factor, on MBEC barrier integrity. The addition of microglia or ATP led to the disruption of the MBEC monolayer and significantly decreased barrier function as measured by trans-endothelial electrical resistance (TEER) and electric cell-substrate impedance sensing (ECIS). Furthermore, ATP promoted the migration of microglia but not macrophages across the MBEC monolayer. An inhibitor of matrix metalloproteinases (MMPs) decreased the transmigration of microglia in our system, indicating that MMPs play a role in microglial chemotaxis. We specifically identify a role for microglia-derived MMP-2. In conclusion, we offer evidence that microglia migration across the brain endothelial cell monolayer is increased in the presence of ATP in a manner that involves MMP secretion.