© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2224-2238 doi:10.1242/jcs.185215 RESEARCH ARTICLE SEPT8 modulates β-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1 Kaisa M. A. Kurkinen1,*, Mikael Marttinen1,*, Laura Turner2, Teemu Natunen1, Petra Mäkinen1, Fanni Haapalinna3, Timo Sarajärvi1, Sami Gabbouj1, Mitja Kurki4, Jussi Paananen4, Anne M. Koivisto3, Tuomas Rauramaa5, Ville Leinonen4, Heikki Tanila6, Hilkka Soininen3, Fiona R. Lucas2, Annakaisa Haapasalo3,6,‡ and Mikko Hiltunen1,3,‡ ABSTRACT a large number of neuritic plaques and neurofibrillary tangles (NFTs) in the neocortex of the brain are detected in Alzheimer’sdisease Dysfunction and loss of synapses are early pathogenic events in ’ patients (Cummings, 2004; Mandelkow and Mandelkow, 1998). Alzheimer s disease. A central step in the generation of toxic amyloid- These consist of amyloid-β (Aβ) peptide and hyperphosphorylated tau β (Aβ) peptides is the cleavage of amyloid precursor protein (APP) by β protein, respectively. It is well established that the increased -site APP-cleaving enzyme (BACE1). Here, we have elucidated β-amyloidogenic processing of amyloid precursor protein (APP), whether downregulation of septin (SEPT) protein family members, leading to the augmented production of Aβ is a key feature underlying which are implicated in synaptic plasticity and vesicular trafficking, the molecular pathogenesis of Alzheimer’s disease (Neve et al., affects APP processing and Aβ generation. SEPT8 was found to 2000). The cleavage of APP by β-site APP-cleaving enzyme 1 reduce soluble APPβ and Aβ levels in neuronal cells through a post- (BACE1) leads to the formation of the N-terminal soluble APPβ translational mechanism leading to decreased levels of BACE1 (sAPPβ) and a membrane-bound APP C-terminal fragment, called protein. In the human temporal cortex, we identified alterations in the C99, which is subsequently cleaved by γ-secretase producing Aβ.By expression of specific SEPT8 transcript variants in a manner that contrast, the majority of APP is cleaved by α-secretases, which leads correlated with Alzheimer’s-disease-related neurofibrillary pathology. to the release of the soluble ectodomain portion of APP (sAPPα)and These changes were associated with altered β-secretase activity. We prevents Aβ formation. Although the role of sAPPα is still under also discovered that the overexpression of a specific Alzheimer’s- debate, recent findings have suggested that the overexpression of disease-associated SEPT8 transcript variant increased the levels of α ’ β sAPP in the brain of Alzheimer s disease model mice overexpressing BACE1 and A peptides in neuronal cells. These changes were human APP with the Swedish mutation and presenilin-1 with deletion related to an increased half-life of BACE1 and the localization of of exon 9 (APP/PS1ΔE9) mitigates synaptic and cognitive defects BACE1 in recycling endosomes. These data suggest that SEPT8 β caused by pre-existing pathology (Fol et al., 2016). modulates -amyloidogenic processing of APP through a mechanism In neurons, BACE1 shows presynaptic localization and it is largely affecting the intracellular sorting and accumulation of BACE1. conveyed in the neuronal recycling endosomes (Cole and Vassar, KEY WORDS: Alzheimer’s disease, Amyloid precursor protein, 2007; Das et al., 2013). BACE1 and APP are segregated in neurons Amyloid-β, BACE1, SEPT8 during resting state conditions, whereas, upon activity-dependent induction, these proteins converge into acidic microdomains INTRODUCTION through an endocytosis-dependent pathway (Das et al., 2013). This Alzheimer’s disease is the most common neurodegenerative disorder is important because BACE1 is optimally active in the acidic in the world, and affects up to 50% of individuals above the age of 85. intracellular compartments, such as early and late endosomes (Kang Owing to the global increase in the aging population, early diagnosis et al., 2012). Based on the amyloid cascade hypothesis, increased and treatment of Alzheimer’s disease are becoming increasingly soluble Aβ peptide levels augment synaptic dysfunction, Ca2+ important from both a human and socioeconomic perspective. dyshomeostasis, inflammation and oxidative stress, as well as tau Alzheimer’s disease is clinically associated with a global cognitive hyperphosphorylation and the formation of NFTs in specific brain decline, and a progressive loss of memory and reasoning. At autopsy, regions in Alzheimer’s disease patients (DeKosky and Scheff, 1990; Hu et al., 2014; Kuperstein et al., 2010). Thus, if started early enough, β 1Institute of Biomedicine, School of Medicine, University of Eastern Finland, 70211 lowering A levels could be sufficient to slow down the disease Kuopio, Finland. 2Eisai Ltd., Bernard Katz Building, University College London, process in Alzheimer’s disease. This idea is further supported by the 3 London WC1E 6BT, UK. Institute of Clinical Medicine – Neurology, School of genetic finding indicating that a naturally occurring A673T rare Medicine, University of Eastern Finland and Department of Neurology, Kuopio University Hospital, 70211 Kuopio, Finland. 4Institute of Clinical Medicine – variant in APP protects from Alzheimer’s-disease- and age-related Neurosurgery, School of Medicine, University of Eastern Finland and Neurosurgery cognitive decline by reducing BACE1-mediated cleavage of APP of NeuroCenter, Kuopio University Hospital, 70211 Kuopio, Finland. 5Institute of β Clinical Medicine – Pathology, School of Medicine, University of Eastern Finland and/or producing less of the aggregation-prone A 40 containing the and Department of Pathology, Kuopio University Hospital, 70211 Kuopio, Finland. A→T variation at position 2 (Di Fede et al., 2009; Jonsson et al., 2012; 6Department of Neurobiology, A.I. Virtanen, Institute for Molecular Sciences, School Benilova et al., 2014; Das et al., 2016). Soluble Aβ42 dimers extracted of Medicine, University of Eastern Finland, 70211 Kuopio, Finland. ’ *These authors contributed equally to this work from the Alzheimer s disease brain have been shown to specifically increase the phosphorylation of tau and subsequently promote ‡ Author for correspondence ([email protected]; [email protected]) neurodegeneration, supporting the intimate link between Aβ and tau ’ M.H., 0000-0003-3566-4096 in Alzheimer s disease pathogenesis (Shankar et al., 2008). This observation also concurs with the notion that synaptic dysfunction is Received 23 December 2015; Accepted 11 April 2016 the best correlate of the cognitive decline in Alzheimer’s disease, Journal of Cell Science 2224 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2224-2238 doi:10.1242/jcs.185215 Fig. 1. See next page for legend. emphasizing the importance of understanding the early mechanisms predictive biomarkers in Alzheimer’s disease, it is necessary to focus leading to synaptotoxicity (Coleman and Yao, 2003). Collectively, in on the cellular processes involved in the early steps of Alzheimer’s order to identify factors that could be utilized as novel drug targets or disease pathogenesis. Journal of Cell Science 2225 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2224-2238 doi:10.1242/jcs.185215 Fig. 1. RNAi screening of septin family member proteins in HEK293-AP– members are known to play a role in the regulation of presynaptic APP cells identifies SEPT8 as a target affecting APP processing and activity and function (Amin et al., 2008; Ito et al., 2009), we β β A 40 and A 42 levels. (A) qRT-PCR was conducted to assess the studied the effects of downregulation of septin family members knockdown effect of 5 mM siRNAs for each target after 72 h of transfection in HEK293-AP–APP cells. GAPDH-normalized mRNA levels of SEPT5 (n=4), (SEPT5, SEPT7, SEPT8 and SEPT10) on APP processing by SEPT7 (n=4), SEPT8 (siSEPT8_2, n=3) and SEPT10 (n=4) in siSEPT- and assessing the levels of total soluble APP and Aβ in human siControl-transfected cells are presented. (B) The levels of total soluble HEK293 cells overexpressing alkaline-phosphatase-conjugated alkaline-phosphatase-linked APP (sAP–APP) in each target siRNA- APP (HEK293-AP–APP) (Lichtenthaler et al., 2003). Validated transfected cell line was normalized to the cell survival (white bars) in each small interfering RNAs (siRNAs) for BACE1 and ADAM17 were sample and is shown as the percentage of the level compared to that with used as positive controls in the screening of total soluble AP–APP siControl. Cell survival was not affected due to the downregulation of septins. (sAP–APP) and Aβ levels. Downregulation of SEPT5, SEPT7 and siControl, siSEPT5, siSEPT7, siSEPT10, siBACE1 and siADAM17 (n=4), and – siSEPT8 (siSEPT8_2, n=3). (C) Three different SEPT8 siRNAs significantly SEPT8 (Fig. 1A) significantly decreased sAP APP levels in the decreased the mRNA levels of SEPT8 in HEK293-AP–APP cells (left). The cell culture medium when normalized to the cell survival levels of Aβ40 and Aβ42 in the cell culture medium are normalized to APP total (Fig. 1B). In addition, a trend towards decreased Aβ40 and protein levels in the respective cell lysates and shown as a percentage of the Aβ42 levels was observed in culture medium samples from cells in level compared to that with siControl (right). n=4. (D) Three different SEPT8 which SEPT5 and SEPT8 was downregulated (Fig. S1A). siRNAs significantly decreased SEPT8 mRNA levels in SH-SY5Y cells stably Conversely, a trend towards increased Aβ40 and Aβ42 levels overexpressing the human APP751 isoform (SH-SY5Y-APP751) (left). The levels of Aβ40 and Aβ42 in the cell culture medium are normalized to the total was observed in cells with SEPT7 and SEPT10 downregulation protein levels of APP in the respective cell lysates and shown as a percentage (Fig. S1A). To further elucidate the SEPT8 findings, two other of the level compared to that with siControl (right). n=4. In A–D, n values are siRNAs targeting different parts of SEPT8 mRNA sequence were biological replicates and results are mean±s.d. *P<0.05, **P<0.01 also applied to the HEK293-AP–APP cells to exclude potential (independent sample t-test).