Chapter II Experimental studies 52 Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration 53 Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration 2.1.1. Regulation of cholesterol biosynthesis via SREBP2 1. Introduction Cholesterol biosynthesis is a tightly regulated biochemical reaction which engages a variety of enzymes and subcellular organelles (Reinhart et al. 1987). It responds rapidly upon the slight alteration in the environment to maintain cholesterol homeostasis, which is crucial for membrane fluidity and signal transduction (Bjorkhem et al. 1998; Frank et al. 2008). Previous studies have shown increased absolute level of cholesterol in the hippocampus after excitotoxic injury induced by KA (Ong et al. 2003). This upregulation might be due to elevated cholesterol biosynthesis in this region since little cholesterol could cross the BBB. The regulation of biosynthesis of cholesterol involves various small molecules and protein groups such as INSIGs and SREBPs (DeBose-Boyd 2008), which assist to maintain cholesterol homeostasis upon dysregulation of physiological cholesterol level . In contrast to SREBP-1 being located in neurons of the cortex and hippocampus (Ong et al. 2000), little is known about the distribution of SREBP-2, the key regulator of cholesterol synthesis (Brown and Goldstein 1997) in the brain of healthy conditions or after neurodegeneration. Therefore, this portion of the study was carried out to explore the distribution of SREBP2 in the normal brain, and mRNA changes of both cholesterol biosynthesis regulators, SREBP-2 and INSIG after KA injury. 54 Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration 2. Materials and methods 2.1. Western blot analysis of distribution of SREBP-2 in normal brain Four male Wistar rats of 200 g each were used for this portion of the study. The number of animals was determined to confirm the distribution of SREBP-2 in various areas of the brain. The rats were deeply anesthetized with an intraperitoneal (IP) injection of 0.4 ml (0.2 ml/100 g) ketamine and xylazine cocktail (prepared with 7.5 ml ketamine (75 mg/kg), ml xylazine (10 mg/kg), and 7.5 ml sterile water) and decapitated. The different parts of the brain including the olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus and hypothalamus, brainstem, and cerebellum were then dissected out, and homogenized in 10 volumes of ice-cold buffer containing 0.32 M sucrose, mM Tris-hydrochloric acid (HCl), pH 7.4, mM ethylenediaminetetraacetic acid (EDTA), and 0.25 mM dithiothreitol. After centrifugation at 15,000 g for 30 min, the supernatant was collected and protein concentrations in the preparation were measured using the BioRad protein assay kit (Bio-Rad Laboratories, CA, USA). Total proteins (60 µg) were resolved in 10% sodium dodecyl sulfate (SDS) polyacrylamide gels under reducing conditions and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). Nonspecific binding sites on the PVDF membrane were blocked by incubation 55 Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration with 5% nonfat milk for hr. The PVDF membrane was then incubated overnight with a goat polyclonal antibody to SREBP-2 (1:100 dilution in Tris buffer saline [TBS]) at °C. The antibody to SREBP-2 (Santa Cruz Biotechnology, CA, USA) was raised against the N-terminal of SREBP-2 and is expected to recognize both the precursor and mature form of SREBP-2. After washing with 0.1% Tween-20 TBS (TTBS), the membrane was incubated with horseradish peroxidase conjugated anti-goat immunoglobulin IgG (Amersham) for hr at room temperature. The protein was visualized with an enhanced chemiluminescence kit (PiPCe, Rockford, IL) according to the manufacturer’s instructions. 2.2. KA injection Fourteen rats received intracerebroventricular (ICV) injection of an excitotoxin KA to see statistically significant changes in the lesioned area. Also, uneven number of animals per group was because some of the animals died after KA injury due to the KA neurotoxicity. The morbidity rate was estimated to be [...]... entailed to examine the effect of KA -induced neurodegeneration on cholesterol biosynthesis 75 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration 2.1.2 Cholesterol biosynthesis pathway 1 Introduction As shown above, cholesterol biosynthesis is controlled by several regulating proteins such as SREBP-2 and INSIGs It also involves a range of intermediates and mediating enzymes... (Lee and Ye 2004), which could be found in KA -induced excitotoxic injury One possibility is that KA lesions could result in rapid degradation of INSIG-1 protein as a result of cellular 74 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration stress (Lee and Ye 2004), leading to increased SREBP-2 expression and cholesterol biosynthesis in neurons at the periphery of the lesions... remains activated even when cholesterol level is augmented (Engelking et al 2005) SREBP-2 expression 73 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration is also affected by the ABCG1 cholesterol transport protein (Burgess et al 2008; Tarr and Edwards 2008), one of the key cholesterol efflux pump In this study, the expression of SREBP-2 was also examined after KAinduced... 84 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration precursors lanosterol, desmosterol and 7-dehydrocholesterol at 1 day after KA injection and significantly decreased in precursors at 1 and 2 weeks postKA injection The observations from both experiments suggested that the brain mounts an early acute response to excitotoxic injury by inducing increased cholesterol synthesis... antigen absorbed peptide instead of antibody showed absence of labeling (Fig 3.2B) 62 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration CX HC CPU TH A B Fig.3.2 Distribution of SREBP-2 in the normal rat brain A: Section immunolabeled with antibody to SREBP-2 Dense staining is observed in the hippocampus (HC), while moderate staining is observed in the cerebral neocortex... callosum (Fig 3.3H) 64 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration CA1 CA3 * A B * DG C CX D PIR E CPU F CCX G CC H Fig.3.3 Light micrographs of SREBP-2 immunolabeled sections from a normal rat brain A: Field CA1 of the hippocampus, labeling in the cell bodies and apical 65 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration dendrites of...Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration 3.2 Immunohistochemistry analyses 3.2.1 Normal rats Dense staining of SREBP-2 was observed in the hippocampus, whereas moderate labeling was observed in the cerebral neocortex and striatum Relatively light labeling was observed in the thalamus, hypothalamus, brainstem and cerebellum (Fig 3.2A) Sections incubated with... normal pattern of staining (Fig 3.6E) Apart from the hippocampus, KA injections also resulted in neuronal damage to the piriform cortex The latter showed generally decreased staining as in the hippocampus (data not shown) Sections incubated with antigen- absorbed antibody showed absence of labeling (Fig 3.6F) 69 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration * *... dendritic shaft (arrows) and in a dendritic spine (arrowhead) C: Asymmetrical synapse (S) between an unlabeled axon terminal containing small round vesicles (AT) and a labeled dendritic spine (arrowhead) Scale: A = 1 µm, B = 0.5 µm, C = 0.2 µm 67 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration 3.2.2 KA injected rats Rats that had been injected with KA for 3 days showed... is lightly stained B: Section from the same animal as A, incubated with antigen-absorbed antibody, showing background staining Scale = 2 mm 63 Chapter 2.1 Changes in cholesterol biosynthesis after KA -induced neurodegeneration Generally, the hippocampus was densely labeled for SREBP-2 The labeling was observed in the cell bodies and apical dendrites of pyramidal neurons, and the neuropil in the stratum . SREBP- 2 in the normal brain, and mRNA changes of both cholesterol biosynthesis regulators, SREBP-2 and INSIG after KA injury. Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration. Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration Chapter 2.1 Changes in cholesterol biosynthesis after KA-induced neurodegeneration. brainstem, and cerebellum (Fig. 3.3G). Little or no staining was observed in white matter tracts including the internal capsule and the corpus callosum (Fig. 3.3H). Chapter 2.1 Changes in cholesterol