Dysregulation of SREBP2 induces BACE1 expression

Abstract

β-Amyloid hyperproduction has been observed in response to alterations in neuronal intracellular cholesterol storage, efflux, and synthesis, induced in rats by a high-fat diet. It has been suggested that cholesterol homeostasis is altered in Alzheimer's disease resulting in higher β- and γ-secretase activity. In the current study the neuronal activation status of sterol regulatory element binding protein 2 (SREBP2) as well as its involvement in β-secretase BACE1 activity was investigated in high-fat fed rats (26% fat and 4% cholesterol for 20 weeks), and in SK-N-BE neuroblastoma cells exposed to 20 μM cholesterol. This work demonstrates that in the brain a hyperlipidic diet is able to induce a hyper-expression of BACE1 and determine an unbalance in cerebral cholesterol homeostasis so that SREBP2 is activated. In addition, we show for the first time the involvement of SREBP2 on expression of BACE1 in SK-N-BE cells exposed to high cholesterol. Although the enhanced risk of Alzheimer's disease in metabolic syndrome is related to several factors, our results suggest that SREBP2, which can be modulated by the impairment of cerebral cholesterol homeostasis, has a direct role on BACE1 expression and may be involved in Alzheimer's disease progression.

Introduction

Recent studies indicate that alterations in cholesterol metabolism induced by a high-fat diet influence some molecular mechanisms involved in neurodegenerative diseases, such as Niemann–Pick, Huntington's disease, prion diseases, and Alzheimer's disease (AD) (Bach et al., 2009, Bi and Liao, 2010, Block et al., 2010, Lütjohann et al., 2000, Solomon et al., 2009), even if controversial data have been reported (Hoyer and Riederer, 2007, Ishii et al., 2003, Zandi et al., 2005). Our study investigates a new potential link between diet-induced alterations in brain cholesterol metabolism and the β-site APP-cleaving enzyme 1 (BACE1) overexpression which represents an early event in AD development.

The CNS contains as much as 23% of total body cholesterol (Dietschy and Turley, 2004) and, since the blood brain barrier (BBB) is nearly impermeable to plasma lipoprotein-associated cholesterol, almost all of brain cholesterol is locally synthesized (Bjorkhem and Meaney, 2004). Excess of cholesterol is exported from the brain in the form of oxysterols which can cross the BBB. The major oxysterol in the brain is the 24S-hydroxycholesterol (24-OH-chol) which regulates genes involved in intracellular free-cholesterol modulation (Wang et al., 2008).

Brain cholesterol synthesis is tightly regulated by a feedback system based on the activity of sterol regulatory element binding protein 2 (SREBP2). In its inactivated form, SREBP2 is retained in the endoplasmic reticulum (ER) by two inhibitory proteins, SCAP and Insig. When cholesterol de novo synthesis is required, SREBP2 is cleaved and translocated to the nucleus, where it activates target genes involved in cholesterol synthesis and uptake (Bengoechea-Alonso and Ericsson, 2007, Goldstein et al., 2006). In contrast, when the intracellular levels of cholesterol increase, SREBP2 is down-regulated. Thus, a proper SREBP2 intracellular traffic is crucial for its activity. Indeed, an up-regulation of SREBP2 in prion-infected neuroblastoma cells has been recently reported. In this study authors suggest that changes in intracellular cholesterol distribution by prion-infection lead to its local decrease at the level of ER and this causes an activation of SREBP2 and cholesterol biosynthesis (Bach et al., 2009). SREBP2 controls the expression of enzymes involved in cholesterol synthesis. One of the target genes activated by SREBP2 is that encoding for the enzyme hydroxy-methylglutaryl-Coenzyme A reductase (HMG-CoA-R), the rate limiting enzyme in the process of cholesterol synthesis (Bengoechea-Alonso and Ericsson, 2007).

Intracellular cholesterol dynamics can be influenced by several factors and a high fat diet is among these. High cholesterol diet leads to metabolic disturbances including obesity, insulin resistance and dislipidemia and may contribute, at least in part, to the development of brain diseases through the action of key mediators such as trophic factors support alterations, mitochondrial abnormalities, oxidative stress increase, and intracellular cholesterol homeostasis failure (Koudinov and Koudinova, 2005, Zhang et al., 2009). Moreover, several studies report that alterations in both plasma and in brain cholesterol levels are related to AD (Kivipelto and Solomon, 2006, Puglielli et al., 2003, Simons et al., 2001, Solomon et al., 2009). Other studies, demonstrating a possible beneficial effect of cholesterol lowering drugs, such as statins or 7-dehydrocholesterol reductase inhibitors, against AD development, support this hypothesis (Kirsch et al., 2003, Parsons et al., 2007, Tong et al., 2009). Indeed, in hypercholesterolemic patients, statins reduce serum β-amyloid peptide (Aβ) levels (Buxbaum et al., 2002, Friedhoff et al., 2001), even if other data are conflicting (Ishii et al., 2003, Serrano-Pozo et al., 2010). However, a recent therapeutic approach to ameliorate amyloid pathology has proposed to act on cholesterol synthesis or on inhibition of the activity of specific enzymes correlated to conversion between free cholesterol and cholesterol esters (Bryleva et al., 2010, Parsons et al., 2007).

The abnormal generation and deposition of Aβ are the pathologic hallmark of AD. Aβ is generated by two sequential proteolytic cleavage steps from the β-amyloid precursor protein (βAPP) (Haass, 2004) that is initially cleaved by the β-secretase BACE1, which has been identified as a β-site APP-cleaving transmembrane aspartic protease. This cleavage is followed by the subsequent proteolysis of the membrane-bound C-terminal fragment catalyzed by the γ-secretase complex (Vassar, 2001). Hypercholesterolemia is linked to increased Aβ production and deposition in the brain both in humans (Kivipelto and Solomon, 2006, Pappolla et al., 2003) and in animals (Ghribi et al., 2006, Thirumangalakudi et al., 2008) and is linked to an increased risk of developing AD (Rushworth and Hooper, 2010). Moreover, Aβ hyperproduction has been observed in response to alterations in neuronal intracellular cholesterol storage, efflux, trafficking, and synthesis, induced in rats by a high-fat diet (Koudinov and Koudinova, 2005) and it has been suggested that cholesterol homeostasis is altered in AD brains resulting in higher β- and γ-secretase activity (Xiong et al., 2008).

In the current study the neuronal activation status of SREBP2 as well as its involvement in BACE1 activity was investigated in in vivo experimental conditions mimicking a cholesterol-rich Western diet feeding and in high cholesterol-exposed SK-N-BE neuroblastoma cells.