Insulin-signaling Pathway Regulates the Degradation of Amyloid β-protein via Astrocytes

Accepted Manuscript Research Article Insulin-signaling pathway regulates the degradation of amyloid β-protein via astrocytes Naoki Yamamoto, Ryo Ishikuro, Mamoru Tanida, Kenji Suzuki, Yuri IkedaMatsuo, Kazuya Sobue PII: DOI: Reference:

S0306-4522(18)30429-9 https://doi.org/10.1016/j.neuroscience.2018.06.018 NSC 18507

To appear in:

Neuroscience

Received Date: Accepted Date:

11 February 2018 11 June 2018

Please cite this article as: N. Yamamoto, R. Ishikuro, M. Tanida, K. Suzuki, Y. Ikeda-Matsuo, K. Sobue, Insulinsignaling pathway regulates the degradation of amyloid β-protein via astrocytes, Neuroscience (2018), doi: https:// doi.org/10.1016/j.neuroscience.2018.06.018

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Insulin-signaling pathway regulates the degradation of amyloid β-protein via astrocytes

Naoki Yamamoto,a, * Ryo Ishikuro,a Mamoru Tanida,b Kenji Suzuki,c Yuri Ikeda-Matsuo,a and Kazuya Sobued

a

Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa 920-1181, Japan

b

c

Department of Physiology II, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan

Laboratory of Molecular Medicinal Science, Department of Pharmacy, College of Pharmaceutical

Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan d

Department of Anesthesiology and Medical Crisis Management, Nagoya City University Graduate

School of Medical Sciences, Nagoya City, Aichi 467-8622, Japan

Running title: Insulin facilitates Aβ degradation of astrocytes

*Corresponding author: Naoki Yamamoto, Ph.D Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa, Japan 920-1181 Phone: 81-76-229-6214 Fax: 81-76-229-6214 E-mail address: [email protected]

Keywords: Alzheimer’s disease (AD), Diabetes mellitus (DM), insulin, amyloid-β (Aβ), astrocytes, neprilysin (NEP), insulin degrading enzyme (IDE), protein metabolism 1

Abbreviations:

AD,

Alzheimer’s

disease;

Aß,

amyloid

ß-protein;

ERK,

extracellular

signal-regulated kinase; IDE, insulin-degrading enzyme; NEP, neprilysin; PI3K, phosphoinositide 3-kinase.

2

Abstract Alzheimer’s disease (AD) has been considered as a metabolic dysfunction disease associated with impaired insulin signalling. Determining the mechanisms underlying insulin signalling dysfunction and resistance in AD will be important for its treatment. Impaired clearance of amyloid-β peptide (Aβ) significantly contributes to amyloid accumulation, which is typically observed in the brain of AD patients. Reduced expression of important Aβ-degrading enzymes in the brain, such as neprilysin (NEP) and insulin-degrading enzyme (IDE), can promote Aβ deposition in sporadic late-onset AD patients. Here, we investigated whether insulin regulates the degradation of Aβ by inducing expression of NEP and IDE in cultured astrocytes. Treatment of astrocytes with insulin significantly reduced cellular NEP levels, but increased IDE expression. The effects of insulin on the expression of NEP and IDE involved activation of an ERK-mediated pathway. The reduction in cellular NEP levels was associated with NEP secretion into the culture medium, whereas IDE was increased in the cell membranes. Moreover, insulin-treated astrocytes significantly facilitated the degradation of exogenous Aβ within the culture medium. Interestingly, pretreatment of astrocytes with an ERK inhibitor prior to insulin exposure markedly inhibited insulin-induced degradation of Aβ. These results suggest that insulin exposure enhanced Aβ degradation via an increase in NEP secretion and IDE expression in astrocytes, via activation of the ERK-mediated pathway. The inhibition of insulin signalling pathways delayed Aβ degradation by attenuating alterations in NEP and IDE levels and competition with insulin and Aβ. Our results provide further insight into the pathological relevance of insulin resistance in AD development.

3

INTRODUCTION Epidemiological studies have revealed a relationship between type 2 diabetes mellitus (T2DM) and an increased risk for Alzheimer’s disease (AD) with age (Akomolafe et al., 2006; Biessels et al., 2006; Sims-Robinson et al., 2010). T2DM-mediated insulin dysregulation is related to AD via a mechanism that promotes the accumulation of hyper-phosphorylated tau, one of the pathological hallmarks of AD (El Khoury et al., 2014). Additionally, AD is characterised by the pathological accumulation of amyloid-β peptides (Aβ). A double transgenic mouse model combining T2DM and AD displayed peripheral insulin resistance, hyperglycaemia, glucose intolerance, increased Aβ deposition in the brain and tau phosphorylation and reduced insulin levels and signalling (Wijesekara et al., 2017). However, the effect of T2DM and altered insulin signalling on Aβ deposition remains unclear. We have previously reported that ageing and peripheral insulin resistance accelerate the assembly of Aβs by increasing GM1 ganglioside (GM1) clustering in detergent-resistant microdomains on neuronal membranes and showed that treatment with insulin inhibits Aβ assembly by decreasing GM1 expression within these microdomains (Yamamoto et al., 2010; 2012). Aβ peptides form as a product from the cleavage of amyloid precursor protein (O’Brien and Wong, 2011). The abnormal accumulation and aggregation of Aβ observed in AD is thought to predominantly result from an imbalance between its production and its clearance via various pathways, including enzyme-mediated degradation (Selkoe and Hardy, 2016). There are several endogenous enzymes capable of Aβ degradation in vivo, including neprilysin (NEP), insulin-degrading

enzyme

(IDE),

plasmin,

several

matrix

metalloproteases

(MMPs),

endothelin-converting enzyme (ECE) and angiotensin-converting enzyme (ACE) (Iwata et al., 2005). Many of these Aβ-degrading enzymes, including NEP, MMP-9, MMP-2, IDE, ECE and ACE, are expressed in astrocytes (Turner and Nalivaeva, 2007; Leissring, 2008). Studies in 4

transgenic animals have shown that Aβ metabolism in the brain is mainly regulated by the two major peptidases NEP and IDE (Iwata et al., 2001; Farris et al., 2003), which show high expression levels in human and rat astrocytes (Carpentier et al., 2002; Dorfman et al., 2010; Yamamoto et al., 2013). Moreover, human brain studies revealed that NEP mRNA and protein expression were lower in AD-affected brain tissue than in control tissue (Yasojima et al., 2001; Wang et al., 2003). Kakiya et al. showed that the phosphorylation state of the intracellular domain of neprilysin regulates its cell surface activity, and modulation of neprilysin localisation regulates extracellular Aβ levels (Kakiya et al., 2012). An age-related decrease in the Aβ-degrading protease NEP was also observed in the brain of the transgenic Tg2576 AD mouse model, in which strong NEP expression was only observed in astrocytes in the vicinity of Aβ plaques (Apelt et al., 2003; Leal et al., 2006). In a recent study, we reported that ketamine reduced NEP expression, but not IDE, through the non-competitive N-methyl-D-aspartate receptor in cultured astrocytes (Yamamoto et al., 2013). Moreover, we indicated that leptin, hydrophobic statins and epigallocatechin gallate (EGCG) reduced NEP expression, but not IDE, in cultured astrocyte cell membranes via activation of extracellular signal-regulated kinase (ERK) (Yamamoto et al., 2014; 2016; 2017). Astrocytes support neurones to maintain normal brain functions, supplying substrates for chemical signals such as neurotransmitters, growth factors and hormones; astroglial dysfunction has been implicated in various neurodegenerative diseases, such as epilepsy and amyotrophic lateral sclerosis (Yong et al., 1988; Seifert et al., 2006). Uptake of glucose and glutamate by astrocytes has been reported to be impaired in AD (Steele and Robinson, 2012). These results have supported the hypothesis that the ability of reactive astrocytes to degrade Aβ might be overwhelmed in the AD-affected brain, with consequent accumulation and deposition of Aβ. Although it is widely accepted that astrocytes play a prominent role in Aβ clearance, the precise molecular mechanisms underlying this clearance remain unknown. 5

In the present study, we investigated the effects of insulin on the expression of Aβ-degrading enzymes and activation of intracellular insulin signalling systems in primary cultured rat cortical astrocytes. We investigated whether the effects of insulin can be nullified by the addition of an inhibitor for the insulin signal transduction pathway. Moreover, we determined whether insulin treatment regulated Aβ degradation via alteration of astrocytic NEP and IDE expression. In particular, we examined whether insulin-associated Aβ degradation could be attenuated by inhibition of insulin signal transduction pathways.

6

EXPERIMENTAL PROCEDURES Materials Insulin, Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), U0126 and LY294002 were purchased from Wako (Osaka, Japan). Anti-NEP antibody was obtained from Leica Microsystems (Newcastle, UK). Anti-IDE antibody was obtained from Calbiochem (San Diego, CA, USA). Anti-Akt and anti-flotillin-1 antibodies were acquired from BD Biosciences (San Jose, CA, USA). Anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phospho-Akt (Ser473), anti-insulin receptor β (IRβ) and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mem-PERTM eukaryotic membrane protein extraction reagent kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Synthetic Aβ1-40 was purchased from Peptide Institute (Osaka, Japan). Anti-Aβ antibody was obtained from Covance (Princeton, NJ, USA).

Cell culture All animal procedures were performed following protocols approved by the Institutional Animal Care and Use Committee of Hokuriku University (Ishikawa, Japan). Primary cortical astrocyte cultures were prepared, as previously described (Yamamoto et al., 2016), from Sprague Dawley rat embryos at 20 days of gestation, obtained from Japan SLC, Inc. (Shizuoka, Japan). Briefly, astrocytes were suspended and cultured in DMEM supplemented with 10% FBS and plated onto 25-cm2 culture flasks (Corning, NY, USA). After 7 days, astrocytes were trypsinised and sub-cultured in 60- or 90-mm (diameter) culture dishes (Corning). All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cell populations consisted of >95% astrocytes, as determined by immunocytochemical examination using an antibody against the astrocyte-specific anti-glial fibrillary acidic protein. Astrocytes were grown to confluence, and the 7

culture medium was replaced with serum-free DMEM. The cells were pharmacologically treated with insulin, inhibitors or a vehicle control [dimethyl sulfoxide (DMSO)]. To examine the effect of insulin, astrocytes were treated with the indicated insulin concentrations for 48 h and then harvested for analysis. To determine the effect of prior insulin signalling pathway inhibition, astrocytes were incubated with an ERK inhibitor (U0126, 10 µM), PI3K inhibitor (LY294002, 10 µM) or vehicle control for 1 h before subsequent treatment with 100 nM insulin for 48 h. The toxicity of insulin treatment was evaluated at 48 h using a lactate dehydrogenase (LDH) release assay.

SDS-PAGE and western blotting To prepare total protein extracts, cultured astrocytes were washed with ice-cold phosphate buffered saline and then lysed using buffer comprising 5 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% Triton X-100 and Complete™ protease inhibitor cocktail (Roche Molecular Biochemicals, Penzberg, Germany). Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific), and 10 µg of total protein was prepared for SDS-PAGE. Proteins were separated on a 10% (NEP and IDE) or 10%/20% gradient (Akt, ERK1/2 and β-actin) polyacrylamide gel (Wako) and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Milford, MA, USA). Each membrane was blocked with Block-Ace (DS Pharma BioMedical, Osaka, Japan) for 1 h at room temperature (25°C) and subsequently incubated at 4°C overnight with an anti-NEP (1:1000), anti-IDE (1:10000), anti-phospho-Akt (1:2000), anti-Akt (1:2000), anti-phospho-ERK1/2 (1:2000), anti-ERK1/2 (1:2000), anti-IRβ (1:2000), anti-flotillin-1 (1:2000) or anti-β-actin (1:5000) antibody. After incubation with primary antibodies, the membranes were washed and incubated with secondary antibodies conjugated with horseradish peroxidase (1:2000; Cell Signaling Technology) for 1 h at room temperature. The proteins were detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) or Immunostar LD (Wako). 8

Quantitative scanning of the blots was performed using Image J version 1.59.

LDH release assay The LDH release assay was performed on culture medium using an LDH release assay kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI, USA). The degree of LDH release in each sample was determined by measuring absorbance at 490 nm. Background absorbance, as assessed using cell-free wells, was subtracted from the absorbance of each test sample. Absorbances measured from three wells were averaged, and the percentage of LDH released was calculated by dividing the absorbance measured from each test sample following treatment with 1% Triton X-100 to induce the release of intracellular LDH, according to manufacturer’s instructions.

Aβ incubation and its detection Synthetic wild-type Aβ40 was dissolved in a 0.02% ammonia solution at 500 µM. This solution was centrifuged at 540,000 ×g for 3 h using a Himac CS120GXL micro ultracentrifuge (Hitachi, Tokyo, Japan) to remove undissolved peptides, which can act as pre-existing seeds. The supernatant was collected and stored in aliquots at −80°C until use. Immediately before use, the aliquots were thawed and diluted with serum-free DMEM. Astrocytes were pretreated with U0126 (10 µM), LY294002 (10 µM) or vehicle control for 1 h and then incubated with or without 100 nM insulin for 48 h. The insulin-treated astrocytes were then incubated with soluble Aβ1-40 (5 µM) for 12 h. The cell medium was collected after Aβ1-40 incubation, centrifuged for 10 min at 1,000 ×g to exclude cell debris and then analysed via western blotting using an anti-Aβ antibody (6E10, 1:5000).

9

Statistical analysis All data are expressed as mean ± standard deviation (SD) of at least four or five independent experiments and were analysed by one-way analysis of variance (ANOVA) combined with Scheffe’s test for all paired comparisons. A P value <0.05 was considered statistically significant.

10

RESULTS Insulin treatment regulated the expression of NEP and IDE in astrocytes To investigate the effect of insulin on astrocytic NEP and IDE expression, we treated primary rat astrocytes with the indicated concentrations of insulin for 48 h and analysed cellular NEP and IDE expression using western blotting. Treating astrocytes with 30 and 100 nM insulin significantly decreased cellular NEP protein levels, but increased IDE protein levels (Fig. 1A). To evaluate the effect of insulin on NEP and IDE expression over time, we treated astrocytes with 50 nM insulin for various time periods (48 h) before harvesting them for western blot analyses. NEP level was significantly decreased and IDE expression was significantly increased after 24 and 48 h of insulin treatment (Fig. 1B). Insulin receptor β (IRβ) and β-actin (internal control) expressions were similar in all samples (Fig. 1A-C). Furthermore, the viability of astrocytes was not affected by treatment with 100 nM insulin for 48 h (Fig. 1D). ERK-mediated pathway activation mediated insulin-induced alterations in NEP and IDE expression and localisation Insulin signalling at the cell surface activates the Akt- and ERK-mediated pathways via tyrosine phosphorylation of insulin receptor substrate proteins, including the insulin receptor substrate (IRS) family and growth factor bound-2 (Grb2)-associated binder (Gab) family (Saltiel and Kahn, 2001; Daly et al., 2002; Gogg and Smith, 2002; White, 2006). We recently showed that the expression of NEP, but not IDE, was reduced in primary cultured astrocytes upon treatment with leptin, simvastatin or EGCG, with these effects mediated by activation of ERK1/2, but not Akt (Yamamoto et al., 2014; 2016). To determine the signalling pathway mediating alterations in astrocytic Aβ-degrading enzymes in the current study, we examined ERK1/2 and Akt phosphorylation in insulin-treated astrocytes using phospho-specific antibodies. Insulin treatment for 0.5 h markedly induced phosphorylation of both ERK1/2 and Akt (Fig. 2A), whereas total 11

ERK1/2 and total Akt levels remained similar across all samples (Fig. 2A). To determine whether activation of one or both of these pathways mediated insulin-induced NEP and IDE alterations, astrocytes were treated with an ERK inhibitor (U0126) or a PI3K inhibitor (LY294002) for 1 h prior to insulin treatment. Pretreatment with U0126, but not with LY294002, significantly attenuated the ability of insulin to decrease NEP expression and increase IDE expression in cultured astrocytes (Fig. 2B). β-actin expression levels were similar in all samples (Fig. 2). We confirmed the specificity of the two inhibitors for their respective pathway targets in cultured astrocytes; phosphorylation of ERK1/2 and Akt after 2 h of insulin treatment was significantly inhibited by pretreatment with U0126 and LY294002, respectively (Fig. 2C). Furthermore, we examined phosphorylation of ERK1/2 and Akt after 48 h of insulin treatment, a time point at which we observed significant changes in NEP and IDE expression. Increased phosphorylation of Akt after 48 h of insulin treatment was significantly attenuated by pretreatment with LY294002. On the other hand, phosphorylation of ERK1 / 2 after 48 h was similar in all samples (Fig. 2D). To investigate how insulin affects the localisation of NEP and IDE expression in astrocytes, we isolated the membrane-bound protein fraction and collected culture medium from insulin-treated astrocytes. The successful isolation of membrane-bound proteins was confirmed by detection of the membrane marker protein, flotillin-1 (Fig. 3A). The membrane-bound protein fractions of insulin-treated astrocytes showed significantly lower NEP expression and significantly higher IDE expression than control astrocytes (Fig. 3A). Pretreatment with ERK inhibitor U0126 attenuated the effects of insulin on membrane NEP and IDE expression (Fig. 3A). Flotillin-1 expression levels were similar in all samples (Fig. 3A). NEP levels in the culture medium from insulin-treated astrocytes were significantly higher than in the medium from vehicle-treated controls, whereas IDE levels in culture medium were not affected by insulin treatment (Fig. 3B). The insulin-induced release of NEP into the culture medium was inhibited by U0126 (Fig. 3B). 12

Insulin treatment of astrocytes facilitated Aβ degradation through secretion of NEP and increased IDE expression Finally, we examined whether the insulin-induced alterations in NEP and IDE facilitated Aβ degradation in the medium of insulin-treated astrocytes and whether inhibition of the insulin signalling pathway attenuated insulin-induced Aβ degradation. To this end, astrocytes were pretreated for 1 h with U0126 or vehicle control, treated for 48 h with insulin or vehicle control and subsequently exposed to exogenously applied Aβ in the culture medium for 12 h. Following incubation, Aβ levels were significantly lower in the medium of insulin-treated astrocytes than those in the medium of non-insulin-treated controls (Fig. 4A). Intriguingly, pretreatment with U0126 attenuated the ability of insulin-treated astrocytes to promote Aβ degradation in the culture medium. U0126 pretreatment alone had no effect on culture medium Aβ levels (Fig. 4A). Previously, it has been reported that treatment with Aβ decreases the expression of neuronal insulin receptor (Du et al., 2011). We investigated whether insulin receptor expression on astrocytes was affected by Aβ exposure in the present study. However, the astrocytic expression of IRβ did not differ between experimental conditions (Fig. 4B).

13

DISCUSSION The prevalence of T2DM and dementia poses great challenges for public health. Given the potential relationship between T2DM, cognitive decline and dementia, it is important to understand how DM and impaired insulin signalling contribute to AD. In the current study, we found that insulin treatment of cultured rat astrocytes induced secretion or release of NEP into the medium and increased IDE expression at the cellular membrane, with these alterations mediated by activation of the ERK signalling pathway. The alterations in NEP and IDE levels facilitated exogenous Aβ degradation in the medium of insulin-treated astrocytes; however, inhibition of the ERK signalling pathway prior to insulin treatment markedly delayed insulin-induced Aβ degradation. Although the pathogenesis of AD has not been fully elucidated, Aβ accumulation and senile plaque formation is a prominent feature of AD. Imbalances in the production and clearance of Aβ may contribute to its accumulation. Cerebral Aβ clearance depends on local degradation and transport across the blood–brain barrier. Particularly, it is still considered that Aβ metabolism and proteolytic degradation are the major processes involved in Aβ clearance and play a major role in the development of AD (Wang et al., 2006; Gough et al., 2011). NEP is an Aβ-degrading metalloendopeptidase, which has been shown to efficiently degrade both the monomeric and oligomeric forms of Aβ (Aβ40 and Aβ42) (Shirotani et al., 2001; Eckman et al., 2003; El-Amouri et al., 2008). IDE are zinc metalloendopeptidases, particularly involved in the degradation of monomeric Aβ species. However, because insulin is an endogenous substrate for IDE, altered insulin signalling would be predicted by upregulation of IDE. Previous studies have reported that NEP expression in the brain correlates with AD risk and pathology; post-mortem studies suggest that NEP levels are reduced in the ageing brain and in tissue with AD pathology, whereas the IDE expression remains unchanged (Yasojima et al., 2001; Caccamo et al., 2005; Wang et al., 2010). 14

The importance of astrocytes in clearance of Aβ from the brain was revealed by several immunohistochemical and experimental studies that demonstrated the uptake and presence of intracellular Aβ in human astrocytes (Funato et al., 1998; Akiyama et al., 1999; Kurt et al., 1999; Thal et al., 2000; Nuutinen et al., 2007; Nielsen et al., 2010; Nielsen et al., 2009). However, the mechanisms mediating uptake and degradation of Aβ in these cells have not been clearly defined. Astrocytes express various Aβ-degrading enzymes, including NEP, IDE, MMP-9, MMP-2, ECE and ACE (Turner and Nalivaeva, 2007; Leissring, 2008). Interestingly, astrocytes cultured from a transgenic mouse model of AD exhibit loss of their ability to clear extracellular Aβ (Liu et al., 2016). We previously reported that leptin and ketamine reduce Aβ degradation through decreased NEP expression in astrocytes (Yamamoto et al., 2013; 2014) and that leptin reduced NEP secretion into the medium of cultured astrocytes (Yamamoto et al., 2014). Recently, we reported that hydrophobic statins and EGCG facilitated degradation of exogenous Aβ through increased NEP secretion from cultured astrocytes (Yamamoto et al., 2016; 2017). In the present study, we observed that insulin treatment of astrocytes induced NEP secretion and IDE expression, facilitating the degradation of exogenous Aβ in the culture medium. In addition, inhibition of the ERK insulin signalling pathway markedly delayed Aβ clearance in insulin-treated cultured astrocytes. Insulin is the predominant mediator of metabolic homeostasis, regulating both glucose and lipids (Shaham et al., 2008; Cheng et al., 2010). Insulin also regulates development, liver gluconeogenesis, fatty acid synthesis and mitogenesis (Saltiel and Kahn, 2001; Taguchi and White, 2008). In the central nervous system (CNS), the functions of insulin include metabolic, neurotrophic, neuromodulatory and neuroendocrine actions (Gasparini and Xu, 2003). Insulin signals through its cell surface receptor, a tyrosine kinase family receptor that undergoes autophosphorylation and recruits adaptor proteins, such as insulin receptor substrates 1 and 2 (IRS1, IRS2) (White, 2003), to initiate pleotropic actions through diverse signalling pathways. Among 15

these downstream signalling pathways, ERK serves as a prominent convergence point (Cheng et al., 2010). IRS also activates the PI3K signalling pathway, leading to Akt activation (Vadas et al., 2011). In the present study, insulin treatment for 0.5 h markedly activated astrocytic MAP kinase and Akt/PI3 pathways. Our recent studies showed that the effects of hydrophobic statins and EGCG on astrocytic NEP secretion are mediated by activation of ERK (Yamamoto et al., 2016 and 2017). Pretreatment with ERK inhibitor, but not PI3K inhibitor, in the current experiments significantly inhibited the insulin-induced alterations in NEP and IDE expression (Fig. 2B and 3). Our studies suggest that insulin induced the degradation of exogenous Aβ through both NEP secretion from astrocytes and the increase in astrocytic IDE expression, with these effects mediated by activation of the ERK-mediated pathway (Fig. 3 and 4). Healthy CNS insulin signalling should be considered an important factor for preventing AD progression. Loss of robust CNS insulin signalling may account, at least in part, for the fact that type 2 diabetes, which can present with reduced brain insulin (Hu et al., 2013), is an important AD risk factor (Ott et al., 1999; Launer, 2005). Deficiency of IRs in the AD-affected brain leads to insulin resistance in AD neuropathology (Craft, 2012). Insulin has been shown to promote brain Aβ clearance via the MEK–ERK-mediated pathway (Gasparini et al., 2001) and prevent its extracellular accumulation and plaque formation (Watson et al., 2003). Conversely, insulin resistance promotes the formation of Aβ fibrils by inducing GM1 ganglioside cluster formation in the presynaptic membrane (Yamamoto et al., 2012). Insulin resistance and Aβ metabolism are mechanistically related in an interactive manner with respect to AD. Long-term insulin resistance in the peripheral tissues promotes insulin resistance in the brain by suppressing insulin uptake and promoting accumulation of Aβ in the brain (Yang et al., 2017). In turn, elevated Aβ induces the removal of cell surface IRs, promoting insulin resistance (Zhao et al., 2008; De Felice et al., 2009). Moreover, because insulin is a better endogenous substrate of IDE than Aβ, changes in insulin 16

signalling can be inferred by upregulation of IDE. In this study, pretreatment with ERK inhibitor–a situation which may mimic conditions of impaired insulin signalling or insulin resistance in vivo–markedly delayed the clearance of exogenous Aβ from the surrounding medium (Fig. 4). Our results suggested that the inhibition of the insulin signalling pathway in the presence of insulin resulted not only in inhibition of IDE and NEP alterations, but also in competitive inhibition between Aβ and insulin in the degradation of Aβ.

17

CONCLUSION We suggest that insulin promotes release of NEP and increased IDE expression at the cell membrane of astrocytes through activation of the ERK-mediated pathway and elevates degradation of soluble oligomeric and monomeric Aβ in the extracellular fluid in the brain. Additionally, inhibition of the insulin signalling pathway in the presence of insulin causes a marked delay in Aβ degradation by astrocytes. A better understanding of the relationship between insulin and the expression of NEP and IDE may allow mitigation of the effects of NEP and IDE-related brain diseases, including AD. Moreover, a deeper understanding of the complicated mechanisms underlying insulin resistance and hyperinsulinemia in the brain may lead to better management strategies for controlling T2DM so as to reduce the subsequent risk for AD-related neuropathology.

18

Acknowledgements This work was supported by a grant from the Grant-in-Aid for Young Scientists B (25870906), and from The

Specific Research

Fund of Hokuriku

19

University, Japan.

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Kakiya N, Saito T, Nilsson P, Matsuba Y, Tsubuki S, Takei N, Nawa H, Saido TC. (2012) Cell surface expression of the major amyloid-β peptide (Aβ)-degrading enzyme, neprilysin, depends

on

phosphorylation

by

mitogen-activated

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kinase/extracellular

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Figure Legends Fig. 1 Effects of insulin on the expression of neprilysin (NEP) and insulin-degrading enzyme (IDE) in cultured primary rat cortical astrocytes. (A) Dose-response relationships, showing the expression of NEP and IDE in astrocytes treated with insulin at the indicated concentrations for 48 h. (B) Time course of NEP and IDE expression in astrocytes treated with 50 nM insulin. (C) Time course of IRβ expression in astrocytes treated with 50 nM insulin. Insulin-treated astrocytes were harvested at the indicated times, and cell lysates were analysed via western blotting using antibodies against NEP, IDE, IRβ and β-actin. NEP, IDE and IRβexpression levels were determined by densitometric scanning of the blot following incubation with a horseradish peroxidase-conjugated secondary antibody. The band densities are presented as percentages of the control (non-treated astrocytes). (D) The concentrations of insulin used in this study did not induce toxicity in the astrocytes. Toxicity is indicated by the amount of lactate dehydrogenase (LDH) released from primary cultured astrocytes incubated for 48 h with the indicated concentrations of insulin. Each value indicates the percentage of LDH released following treatment with insulin, relative to the LDH released following treatment with Triton X-100. Each column indicates the mean ± standard deviation (SD) of four or six independent experiments. *P < 0.005, **P < 0.0001 [one-way analysis of variance (ANOVA) and Scheffe’s test].

Fig. 2 Contribution of insulin signalling pathway to the expression of neprilysin (NEP) and insulin-degrading enzyme (IDE) in primary cortical rat astrocytes. (A) Astrocytes were treated with 50 nM insulin for 0.5, 1, 2 or 24 h. The lysates from treated cells were processed for western blotting using antibodies specific to ERK, phospho-ERK (pERK), Akt, and phospho-Akt (pAkt). (B) Astrocytes were treated in the presence or absence of 50 nM insulin or vehicle (DMSO) for 48 h following pretreatment with or without ERK inhibitor U0126 (10 µM) or PI3K inhibitor LY294002 28

(10 µM) for 1 h. Activation and inhibition of insulin signalling pathways following inhibitor pretreatment and subsequent insulin treatment. Astrocytes were treated in the presence or absence of 50 nM insulin or vehicle (DMSO) for 2 (C) or 48 h (D), following pretreatment with or without U0126 (10 µM) or LY294002 (10 µM) for 1 h. The treated cell lysates were analysed via western blotting using an anti-NEP, anti-IDE, anti-ERK, anti-pERK, anti-Akt, anti-pAkt or anti-β-actin antibody. The expression levels of NEP, IDE, ERK, pERK, Akt and pAkt were determined by densitometric scanning of the blot following incubation with a horseradish peroxidase-conjugated secondary antibody. The band densities are presented as percentages of the control (non- or vehicle-treated astrocytes) at each timepoint. Each column indicates the mean ± standard deviation (SD) of four or five independent experiments. *P < 0.001 (one-way ANOVA and Scheffe’s test). Con, control; ins, insulin; LY, LY294002.

Fig. 3 Expression of neprilysin (NEP) and insulin-degrading enzyme (IDE) at the cell membrane and in culture medium of insulin-treated astrocytes. Astrocytes were incubated in the presence or absence of 50 nM insulin for 48 h, after pretreatment with or without 10 µM U0126 for 1 h. (A) Membrane fractions were isolated from insulin-treated astrocytes and were analysed via western blotting using an anti-NEP, anti-IDE or anti-flotillin-1 antibody. (B) The culture medium (total protein 30 µg) from insulin-treated astrocytes was analysed via western blotting using an anti-NEP or anti-IDE antibody. NEP and IDE expression levels were determined by densitometric scanning of the blot following incubation with a horseradish peroxidase-conjugated secondary antibody. The band densities are presented as percentages of the control (vehicle-treated astrocytes). Each column indicates the mean ± standard deviation (SD) of five independent experiments. *P < 0.0001 (one-way ANOVA and Scheffe’s test). Con, control; ins, insulin.

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Fig. 4 Effects of insulin on the degradation of exogenous Aβ in cultured astrocyte medium. Astrocytes treated with 50 nM insulin for 48 h were incubated with 5 µM soluble Aβ40 for 12 h. (A) The medium from Aβ40-treated astrocyte cultures was analysed via western blotting using an anti-Aβ antibody (6E10). (B) The treated cell lysates were analysed via western blotting using anti-IRβ antibodies. The levels of Aβ40 and IRβ were determined by densitometric scanning of the blot following incubation with a horseradish peroxidase-conjugated secondary antibody, and band densities are presented as percentages of the control (vehicle-treated astrocytes). Each column indicates the mean ± standard deviation (SD) of five independent experiments. *P < 0.001, **P < 0.0001 (one-way ANOVA and Scheffe’s test).

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Highlights 1. Alzheimer’s disease is associated with impaired insulin signalling. 2. Insulin-treated astrocytes induced neprilysin secretion and IDE expression via activation of the ERK-mediated pathway. 3. Insulin facilitated degradation of exogenous Aβ in the medium of cultured astrocytes. 4. Insulin-mediated facilitation of Aβ degradation was markedly attenuated by inhibition of the ERK-mediated pathway.

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