Down-regulation of Seladin-1 Increases BACE1 Levels and Activity through Enhanced GGA3 Depletion during Apoptosis

Abstract

Seladin-1 is a neuroprotective protein selectively downregulated in brain regions affected in Alzheimer disease (AD).Seladin-1 protects cells against ␤-amyloid (A␤) peptide 42-and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the ␤-secretase (BACE1) function and ␤-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that ϳ60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or A␤ secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized ␥-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced ␤-amyloidogenic processing of APP and ultimately increased A␤ production. These adverse changes associated with decreased cell viability in seladin-1 siRNAtransfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased ␤-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis. Alzheimer disease (AD)2 is the most common neurodegenerative disorder leading to dementia. It is neuropathologically characterized by extracellular amyloid plaques as well as intraneuronal neurofibrillary tangles, composed of ␤-amyloid peptide (A␤) and hyper-phosphorylated Tau, respectively. A␤ is generated from the ␤-amyloid precursor protein (APP) after sequential cleavages by ␤-and ␥-secretases (1). Importantly, mutations in APP and PSEN1 and -2 genes have been shown to increase the production of the 42-amino acid-long A␤42 and to cause the familial autosomal dominant form of AD. Based on this notion, it has been proposed that aberrant metabolism of APP is the initiating event in AD pathogenesis, which is followed by other adverse events, such as inflammation, oxidative stress, and neurofibrillary tangle formation (2). Recently, this model was challenged by a dual pathway hypothesis, which proposed that A␤ and Tau can actually be linked by separate mechanisms driven by common upstream initiators, such as apolipoprotein E (apoE) or glycogen synthase kinase-3 (3). Therefore, considering th...

7,8-Dihydroxyflavone, a Small-Molecule TrkB Agonist, Reverses Memory Deficits and BACE1 Elevation in a Mouse Model of Alzheimer's Disease

“…Multiple mechanisms are recently proposed to mediate the upregulation of BACE1 associated with AD. Those include the increased phosphorylation of the translation initiation factor eIF2a Devi and Ohno, 2010b;O'Connor et al, 2008), caspase-3-dependent inactivation of GGA3 leading to decreased lysosomal degradation of BACE1 (Sarajarvi et al, 2009;Tesco et al, 2007), changes in microRNA expression profiles (Hebert et al, 2008;Wang et al, 2008), p25/cyclin-dependent kinase 5 pathways (Cruz et al, 2006;Wen et al, 2008), calpain activation (Liang et al, 2010), the receptor for advanced glycation end products (RAGE) (Cho et al, 2009;Guglielmotto et al, 2010), and oxidative stress or related signals such as nuclear factor-kB, c-Jun N-terminal kinase, and p38 MAPK (Chen et al, 2011;Chen et al, 2008;Coma et al, 2008;Xiong et al, 2007). Further study will be needed to determine the molecular pathways by which activation of BDNF-TrkB signaling may counteract the BACE1 elevation in 5XFAD mice.…”

Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells

“…regions such as the inferior temporal cortex [52,53], and the enzyme has been shown to protect neurons from Abmediated toxicity and to decrease Ab 42 production by counteracting the b-secretase cleavage of AbPP [54,55]. Moreover, DHCR24-dependent cholesterol synthesis reduces the membrane aggregate interaction and cell damage associated with amyloid treatment [56].…”

Cysteine 27 Variant of the δ-Opioid Receptor Affects Amyloid Precursor Protein Processing through Altered Endocytic Trafficking

“…Primary antibodies against c-Myc (4A6 [Millipore] and A-14 and 9E10 [Santa Cruz]), FLAG (M2; Sigma), APP's C terminus (A8717; Sigma), APP's N terminus (MAB348, clone 22C11; Millipore), secreted N-terminal APP fragments (sAPP␣) (6E10; Biosite), ␤-actin (AC-15; Sigma), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245; Abcam) were used for immunoblotting. Antibody A8717 has previously been shown to specifically detect the APP C-terminal fragments (CTFs) C83, C89, C99, and APP intracellular domain (AICD), while 22C11 and 6E10 are antibodies commonly used to detect total sAPP (sAPPtot) and sAPP␣, respectively (12,16,36). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare), an enhanced-chemiluminescence (ECL) substrate (Amersham Biosciences/GE Healthcare) was applied to the membranes and protein bands were detected with an ImageQuant RT ECL camera (GE Healthcare) or with ECL hyperfilm (GE Healthcare) scanned with a Umax Powerlook 1120 color scanner using the Image Master 2D Platinum 6.0 software.…”

“…The AP ectodomain was fused to the N terminus of full-length APP695, lacking a signal peptide (27). SH-SY5Y human neuroblastoma cells overexpressing the APP751 isoform (SH-SY5Y-APP751) were grown as described previously (36). These cells express approximately 5 times more APP751 and A␤ than naive SH-SY5Y cells (reference 36 and unpublished observations).…”

“…sAPP␣ (secreted N-terminal APP fragments) and sAPPtot (ϭ sAPP␣ ϩ sAPP␤) levels were detected from cell culture media using Western blot analysis with the 6E10 (Signet Laboratories) and 22C11 (Mab348; Millipore) antibodies, respectively. ␤-Amyloid 40 (A␤40) levels in the SH-SY5Y-APP751 cell culture media were determined by using the human A␤40 enzyme-linked immunosorbent assay (ELISA) kit (the Genetics Company) as described previously (36). sAP-APP measurements from HEK293 AP-APP cells were performed as previously described (12).…”