Metab Brain Dis (2012) 27:443–451 DOI 10.1007/s11011-012-9343-5

ORIGINAL PAPER

Effects of statins and cholesterol on memory functions in mice Ravindra M. Ghodke & Nagesh Tour & Kshama Devi

Received: 19 July 2012 / Accepted: 8 October 2012 / Published online: 16 October 2012 # Springer Science+Business Media New York 2012

Abstract Studies on influence of lipid lowering therapies have generated wide controversial results on the role of cholesterol on memory function. However recent studies revealed that cholesterol lowering treatment substantially reduce the risk of dementia. The objectives of this study were to analyze the effect of statins on memory function and to establish the relationship between increase/decrease in cholesterol synthesis, total cholesterol level and memory function in animals. We examined the relationship between biosynthesis of cholesterol and memory function using two statins (lipophilic simvastatin and hydrophilic pravastatin) and high cholesterol diet in mice for 15 days and 4 months. Memory performance was evaluated with two different behavioral tests and various biochemical parameters such as serum cholesterol, whole brain cholesterol, brain 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) activity and brain acetylcholine esterase (AChE) activity. We found that statin treatment for 4 months, but not for 15 days, showed significant improvement in memory function whereas high cholesterol diet showed significant impairment of memory. However long-term statin treatment showed significant decrease in serum cholesterol level as well as brain AChE level. Moreover high cholesterol diet showed significant decrease in memory function with an increase in serum cholesterol level as well as brain AChE level. There is no direct correlation between brain cholesterol level, as well as HMG-CoA activity with memory function regulation. However there is definite link between plasma cholesterol

R. M. Ghodke (*) : K. Devi Al-ameen College of Pharmacy, Banglore, Karnataka 560 027, India e-mail: [email protected] N. Tour K. T. Patil College of Pharmacy, Osmanabad, Maharashtra 413 501, India

level and AChE level. A long-standing plasma cholesterol alteration may be essential to regulate memory function which in turn might be mediated through AChE modulated pathway. Keywords Brain cholesterol . Statins . AChE . Memory function

Introduction Within last few years plasma cholesterol has been linked to Alzheimer’s disease pathology, and epidemiological data suggest that administration of lipid lowering agents such as statins could be of benefit in decelerating the incidence of Alzheimer’s disease. Recent epidemiological studies also indicate that the prevalence of dementia is reduced among patients taking statins over a long period of time (Lutjohann et al. 2004). However, certain case studies performed on human subject’s states that statins may be associated with cognitive impairment. Data from the literature also suggest that memory loss were more associated with lipophilic statins (Wagstaff et al. 2003). It has been found that brain cholesterol synthesis is significantly affected by a short-term treatment with high doses of lipophilic simvastatin but not with hydrophilic pravastatin, however whole-brain cholesterol turnover is not effected (Thelen et al. 2006). Inability of pravastatin to decrease brain cholesterol synthesis might be pertaining to its lower lipophilicity which restricts its entry through blood brain barrier (BBB), moreover one possible reason for unaltered brain cholesterol might be its longer half-life (Andersson et al. 1990). This indicates that a long-term treatment is required to study the influence of cholesterol lowering agents on brain cholesterol and thereby on memory. It is not well understood whether statin-induced neuroprotection is due to simply lowering cholesterol levels or such protection is cholesterol independent (Franke et al. 2007). Besides lowering cholesterol statins were

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found to exhibit multiple effects such as alteration in various neurotransmitter activities, which in turn may contribute in improvement of memory performances (Pereira et al. 2010). Thus it is of interest whether treatment with statins will be able to affect whole brain cholesterol level as well as memory function. In addition treatment with statins for varying duration will give idea on influence of treatment duration on brain cholesterol level and memory. Furthermore, there is a need to study the effects of hydrophilic and lipophilic statins, as well as high cholesterol diet on whole brain cholesterol on memory function. In addition cholesterol levels in the brain and in the blood were found to correlate positively with AChE and possess the potency to increase AChE activity (Cibickova et al. 2007). The current clinical evidence is not strong enough to support the widespread use of statins to treat dementia (Butterfield et al. 2011). Hence the present study was designed to find out the influence of plasma cholesterol level alteration on brain cholesterol biosynthesis, on cholesterol level and AChE level as well as on the memory function.

Methods Animals C57BL/6 mice of either sex, weighing 20–25 g were housed in groups of five in clean polypropylene cages at room temperature (25 °C) and humidity of 45-55 %. 12 h light/ dark cycle was maintained in the animal house. The animals were fed with commercially available standard pellet chow (Amrut Feeds, Bangalore) and had a free access to water. The study was conducted after obtaining Institutional Ethical Committee clearance (AACP/IAEC/M-65/2006). Preparation of drug Pravastatin sodium and simvastatin were provided by Biocon Limited, Bangalore. Pravastatin was dissolved in distilled water and simvastatin was suspended in distilled water using 2 % (v/v) of Tween 80 as a suspending agent. Mice received 20 mg/kg of simvastatin or 25 mg/kg pravastatin by oral gavage once daily. Groups Mice of either sex were randomly divided into 6 groups of 12 animals each. In each group number of male and female mice was equal. Group I: Age matched control Group II: High cholesterol treated (120 Days) Group III: Short term simvastatin treated (15 Days)

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Group IV: Short term pravastatin treated (15 Days) Group V: Long term simvastatin treated (120 Days) Group VI: Long term pravastatin treated (120 Days) Memory function evaluation Six mice from each group were used in assessment of memory, whereas remaining 6 mice were used for the measurement of serum cholesterol, total brain cholesterol, AChE activity and HMG CoA reductase activity. Behavior tests were performed at the end of respective treatment duration. Elevated plus maze test On the 1st day, the training was carried out by placing mice individually at the end of one open arm facing away from the central platform and the time it took for the mouse to move from the open arm to either of the enclosed arms (transfer latency) was recorded. Transfer latency was the time elapsed between the time when the animal was placed in the open arm and the time when its four legs crossed the imaginary line in the middle of the enclosed arm. In this experiment, if the mouse did not enter the enclosed arm within 90 s, it was pushed gently on the back into the enclosed arm and the transfer latency assigned to 90 s. The mouse was allowed to move freely in the plus-maze regardless of open and closed arms for 10 s after the measurement of transfer latency. The mouse was then gently taken out of the plus-maze and was returned to its home cage. The maze was cleaned with an alcohol-water solution after each mouse to remove olfactory cues. Twenty-four hours later, the retention test was performed. The mice were again put into the elevated plus-maze and transfer latency was again recorded. If the mouse did not enter the enclosed arm within 90 s, the transfer latency was assigned to 90 s (Itoh et al. 1991). Step down passive avoidance test Mice were placed on the platform individually and their latency to step down on the grid with all four paws was measured. In training sessions, immediately after stepping down on the grid, the mice received a 2.0 s scrambled foot shock. The shock intensity was 0.4 mA. Mice are then removed from the apparatus and returned to home cage. The apparatus was cleaned with alcohol-water mixture in order to remove any olfactory cues. Animals were given a single training session, followed by a retention test session 24 h later. In test sessions, no foot shock was administered and the step-down latency (maximum 180 s) was used as a measure of retention (Quevedo et al. 1999).

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Estimation of serum cholesterol Total cholesterol was estimated by using Enzokit Cholesterol (CHOD-PAP) from Ranbaxy Fine Chemicals Limited (RFCL) Diagnostics Division. Estimations of total cholesterol, acetyl cholinesterase activity and HMG CoA reductase activity in whole brain Tissue preparation At the end of a respective treatment duration the mice were decapitated, brains were dissected out. Each whole brain was weight recorded and divided into three equal portions after proper mincing. One portion each is used for total cholesterol estimation, AchE activity and HMG CoA activity measurement. Brain cholesterol The brain tissue was homogenized with 2:1 chloroformmethanol mixture (v/v) to a final 20-fold dilution of the volume of tissue sample. The tube of homogenizer was calibrated at the volume of final dilution of the particular tissue homogenate. Each tissue was homogenized for 3 min in order to get a uniform homogenate. Volume is adjusted up to the calibration mark with chloroform-methanol mixture and the homogenate is filtered through a fat-free paper in to glass stoppered vessel. The crude extract was mixed thoroughly with 0.2 its volume of 0.29 % sodium chloride solution and the mixture was allowed to separate into two phases, without interfacial fluff by centrifugation for 10 min at 2400 rpm. As much of the upper phase as possible was removed by suction. Removal of solutes of the interface was completed by rinsing it three times with small amounts of pure solvents upper phase in such a way that as not to disturb the lower phase. Finally, the lower phase and remaining rinsing fluid were made into one phase by the addition of methanol (Bradford et al. 1991). A 0.4 ml of brain extract as well as each dilution of stock cholesterol was placed individually in test tubes and diluted with 6.0 ml of the FeSO4 reagent. A 2.0 ml portion of H2SO4 was blown into the mixture with force to obtain immediate uniformity. After 10 min. absorbance is measured using UV spectrophotometer at 490 nm. All the readings were made against blank prepared with 0.4 ml of chloroform methanol instead of tissue extract. A graph of standard cholesterol versus absorbance was plotted. The amount of cholesterol present in brain extract was determined with the help of standard cholesterol graph. Estimation of acetyl cholinesterase enzyme activity of mice whole brain The tissue was homogenized (approximately 20 mg of tissue per ml of phosphate buffer, pH 8.0, 0.1 M). A 0.4 ml aliquot

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of this homogenate was added to a cuvette containing 2.6 ml of phosphate buffer (pH 8.0, 0.1 M). 100 μl of the DTNB reagent was added and the absorbance was measured at 412 nm, when this had stopped increasing the absorbance was set to zero. In other cuvette, 0.4 ml aliquot of homogenate, 2.6 ml of phosphate buffer (pH 8.0, 0.1 M), 100 μl of DTNB reagent and the substrate, acetylthiocholine iodide 20 μl were added. Changes in absorbance were recorded and the change in absorbance per min was calculated (Ellman et al. 1961). The rate in moles of the substrate hydrolyzed per minute per gram of tissue is calculated by R


ΔA ΔA 1  ¼ 5:74 104 1:36ð104 Þ ð400=3120ÞC0 C0 Where,

R ΔA C0

rate, in moles substrate hydrolyzed per min per g of tissue. change in absorbance per min original concentration of tissue (mg/ml).

Statistical analysis Results are reported as mean±standard error of the mean (SEM). The result data of control group with treated group was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. The data of transfer latency (TL) and Step down latency (SDL) of training session (Day 1) with retention session (Day 2) was analyzed by unpaired T-test. The unpaired t method tests the null hypothesis that the population means related to two independent, random samples from an approximately normal distribution are equal. The statistical analysis was carried out by SPSS 15 software.

Results Elevated plus maze test Short term treatment (15 days) Transfer latency (TL) was measured during training session (day 1) and during retention session performed 24 h after the training session (day 2). It was observed that TL of pravastatin treated group and simvastatin treated group were decreased when compared with TL of age matched control group during training as well as retention session (Table 1). Decrease in TL of retention session (Day 2) was observed when compared to TL of training session (day 1) in pravastatin as well as simvastatin treated group.

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Table 1 Effect of short-term treatment with simvastatin and pravastatin on transfer latency(s) to the enclosed arm of the elevated plus-maze Test session

Control

Simvastatin

Pravastatin

Training (Day1) Retention (Day2)

39.67±4.66 30.17±4.31

32.00±2.62 29.17±3.51

31.68±1.66 29.83±3.49

Values are expressed as Mean±SEM, statistical analysis was carried out by unpaired T-test for comparison of TL during Day 1 with Day 2 and by ANOVA followed by Dunnet’s test for comparison of treated groups with control group

Long term treatment (4 months) In comparison with the age matched control group, TL on day 1 was decreased in simvastatin treated group and pravastatin treated group. While in high cholesterol diet fed group, increase in TL when compared with the age matched control group and simvastatin and pravastatin treated group was found to be insignificant. On day 2, TL was significantly decreased in simvastatin as well as in pravastatin treated group, whereas TL was increased significantly in high cholesterol diet treated group when compared with the age matched control group and simvastatin and pravastatin treated group. TL measured during the training session (day 1) was compared with TL during retention session (day 2), it was found that, TL was significantly shortened during the retention session when compared with TL measured during the training session in both simvastatin treated and pravastatin treated group, while the high cholesterol treated group did not show significant difference (Table 2).

measured during training session (day 1) and retention session (day 2), showed significant increase in SDL measured on day 2 as compared to SDL on day 1. However difference in significance level was observed between age matched control group and both simvastatin treated group and pravastatin treated group (Table 3). Long term treatment (4 months) During the training session of step down type passive avoidance task, simvastatin treated and pravastatin treated groups showed slightly increase in SDL compared to age matched control group. Whereas high cholesterol diet fed group showed decrease in SDL compared to age matched control group. However, during the retention session performed 24 h after the training session high cholesterol diet fed group showed decrease in SDL as compared to that of age matched control group. Whereas simvastatin treated group as well as pravastatin treated group significantly prolonged SDL. When SDL measured during training session (day 1) was compared with SDL measured during retention session (day 2), it was found that all groups showed significant increase in SDL measured on day 2 as compared to SDL measured on day 1. However difference in significance level was observed between age matched control group, high cholesterol diet fed group and both simvastatin and pravastatin treated groups (Table 4). Estimation of serum cholesterol, brain cholesterol, brain HMG-CoA reductase activity and brain AChE activity

Step down passive avoidance test

Serum cholesterol

Short-term treatment (15 days)

At the end of respective treatment durations serum cholesterol was found reduced significantly in the animals treated with simvastatin for a short-term treatment (15 days), as well as for long term treatment (4 months) (Table 5). In the animals treated with pravastatin, serum cholesterol was found to be reduced significantly after a short-term treatment as well as after a long-term treatment (Table 5). Whereas the animals treated with high cholesterol diet exhibited significantly high level of serum cholesterol.

Step down latency (SDL) was measured during training session (day 1) and during retention session performed 24 h after the training session (day 2). It was observed that there was slightly increase in SDL of pravastatin treated group as compared to simvastatin treated group and SDL of age matched control group during training as well as retention session. The comparison of SDL of all groups

Table 2 Effect of long-term treatment with high cholesterol diet, simvastatin and pravastatin on transfer latency(s) to the enclosed arm of the elevated plus-maze Test session

Control

High cholesterol

Simvastatin

Training (Day1)

39.67±4.66

49.33±4.47

18.83±1.58##

Retention (Day2)

30.17±4.31

45.83±4.38

##

Pravastatin 16.50±2.11### ##

10.17±1.62**,

11.00±2.11*,##

Values are expressed as Mean± SEM, statistical analysis was carried out by unpaired T-test for comparison of TL during Day 1 with Day 2 and by ANOVA followed by Dunnet’s test for comparison of treated groups with control group. *p<0.05, **p<0.01 versus Day 1 and ## p<0.01, ### p< 0.001 versus control group

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Table 3 Effect of short-term treatment with simvastatin and pravastatin on step down latency(s) of the step down passive avoidance test

Discussion

Test session

Cognitive impairment in elderly people, once called senile dementia affects more than 10 % population older than 65 years. Lipid related mechanisms are thought to have a role in the pathogenesis of dementia (Jick et al. 2000). Several clinical and epidemiological studies suggest elderly individuals with elevated plasma cholesterol have increased susceptibility to learning and memory disorders (Refolo et al. 2001). In addition, lipid-lowering agents like statins have been reported to substantially reduce the risk of dementia in the elderly (Jick et al. 2000). Perversely, certain studies raise the possibility that greater serum concentrations of LDL-C were positively associated with better memory performance (Henderson et al. 2003). These intriguing findings raised the necessity to establish the relationship between memory function and cholesterol level. Better understanding of the effects of statins on brain metabolism becomes more important because many studies bring evidence of a possible link between cholesterol and neurodegeneration (Cibickova 2011). The ability of certain lipid lowering agents to permeate through the BBB seems to be related to their higher lipophilicity. The brain cholesterol synthesis in mice is affected by high dose of simvastatin but not of pravastatin (Thelen et al. 2006) and hence we studied a hydrophilic (pravastatin) and a lipophilic statin (simvastatin) to find out whether the memory function alteration by statins is dependent on lipophilicity or on peripheral cholesterol regulation. There are reports which state that whole-brain turnover is not disturbed by short-term treatment with cholesterol lowering agents (Lutjohann et al. 2004; Thelen et al. 2006). This can be justified with the previous findings that the estimated half life of cholesterol has been found to be 4 to 6 months in rats (Andersson et al. 1990). For finding the influence of statin treatment duration on memory function and on brain cholesterol level we examined short-term as well as longterm treatment effects of statins. The most important factors regulating cholesterol level in the brain are the rate of local synthesis and the rate of efflux of cholesterol (Lutjohann et al. 2004). An enzyme HMGCoA reductase is responsible for the conversion of HMG-

Control

Simvastatin

Pravastatin

Training (Day1) 5.17±1.40 5.67±1.5 5.83±1.47 Retention (Day2) 50.83±10.34** 51.83±7.36*** 55.83±4.95*** Values are expressed as Mean±SEM, statistical analysis was carried out by unpaired T-test for comparison of SDL during Day 1 with Day 2 and by ANOVA followed by Dunnet’s test for comparison of treated groups with control group. **p<0.01, ***p<0.00 versus Day 1

Brain cholesterol Mice treated for 4 months with simvastatin showed significant decrease in brain cholesterol level as compared to control group. Mice with 4 months treatment of high cholesterol diet showed significant increase in brain cholesterol level as compared to control group (Table 6). However mice with 15 days treatment of simvastatin and mice with 15 days as well as 4 months of treatment with pravastatin failed to significantly alter brain cholesterol level. Brain HMG CoA reductase activity Treatment with simvastatin for short duration as well as long duration resulted in significant up-regulation of HMG-CoA reductase activity in mice brain as compared to control. Treatment with high cholesterol diet showed significant down-regulation of HMG-CoA reductase activity. However, in groups treated with pravastatin for a short duration as well as long duration, there was no significant change in HMGCoA reductase activity (Tables 5 and 6). Brain AChE reductase level Brain AChE level was found to be decreased after treatment with simvastatin, and pravastatin for short term treatment (Table 5) as well as long term treatment (Table 6) as compared to age matched control. Moreover, treatment with high cholesterol diet significantly increased brain AChE level (Table 6).

Table 4 Effect of long-term treatment with high cholesterol diet, simvastatin and pravastatin on step down latency(s) of the step down passive avoidance test Test session

Control

High cholesterol

Simvastatin

Pravastatin

Training (Day1)

5.2±1.4

4.3±0.7

5.0±0.8

5.7±1.1

Retention (Day2)

50.8±10.3**

10.5±2.5*

134.0±15.3***,###

103.5±15.8***,#

Values are expressed as Mean±SEM, statistical analysis was carried out by unpaired T-test for comparison of SDL during Day 1 with Day 2 and by ANOVA followed by Dunnet’s test for comparison of treated groups with control group. *p<0.05, **p< 0.01, ***p<0.001 versus Day 1 and # p<0.05, ### p< 0.001 versus control group

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Table 5 Serum cholesterol, brain total cholesterol, AChE activity and HMG CoA reductase activity in mice brain, after short-term treatment Parameter

Control

Simvastatin

Pravastatin

Serum cholesterol (mg/dl) Brain cholesterol (mg/g wet tissue) Activity of brain HMG CoA R (nmoles of mevalonate /min/mg protein) Activity of brain AchE (μ moles of substrate hydrolyzed/min)

92.43±6.08 15.20±0.33 0.218±0.010 6.78±0.10

56.42±7.25* 15.01±0.29 0.295±0.016 6.11±0.16**

66.94±4.41* 15.13±0.41 0.224±0.010 6.19±0.11**

Values are expressed as Mean±SEM, statistical analysis was carried out by ANOVA followed by Dunnett's test. *p<0.05, **p<0.01, versus control group

CoA to mevalonate, which is a rate limiting step in cholesterol biosynthesis cascade (Thelen et al. 2006). The decrease in HMG-CoA reductase blocks long term potentiation (LTP) in mice, which is required during learning and memory storage (Kotti et al. 2006). On the contrary, inhibition of HMG-CoA reductase shows improvement in functional performance in mice (Wang et al. 2007). To evaluate brain cholesterol synthesis, we performed estimation of HMG-CoA reductase activity. The modulation of learning and memory involves many typical neurotransmitters such as acetylcholine, amino acids and serotonin. Among these, acetylcholine is an important neurotransmitter critical for learning and memory, and loss of cholinergic function contributes to the profound learning and memory deficits associated with age-related dementia and Alzheimer’s disease (Yan-qiang et al. 2007). However, diet may also influence the synthesis of AChE and decreased levels of AChE in hippocampal and cortical region but not cerebellar region have been demonstrated in animal models receiving high-fat diet (Lane and Farlow 2005). On the contrary cholesterol levels in the brain and in the blood were found to correlate positively with AChE and possesse the potency to increase AChE activity (Yan-qiang et al. 2007; Lane and Farlow 2005). Therefore, to establish whether statin treatment for varying duration affect the AChE level, which may be responsible for the observed alterations in memory function, we estimated the AChE level. Effect of cholesterol on learning and memory Long-term treatment with simvastatin as well as pravastatin showed improvement in memory retention in both the learning

and memory models. However, short-term treatment failed to show any significant improvement in memory retention. High cholesterol diet showed significant decrease in memory retention during one of the behavioral test. This memory impairing effect of high cholesterol diet is supported by previous studies showing decrease in memory performance after feeding mice with high lipid diet (Hoglund et al. 2004). The statin treatment significantly improved memory in mice. In fact, statins have multiple effects besides lowering cholesterol, which may contribute to alteration in memory performance such as inhibition of dimerization of β-secretase (Parsons et al. 2006), alterations in multiple gene expression patterns (Johnson-Anuna et al. 2005), and alteration in the activity of various neurotransmitters. Statins-induced disturbances in the equilibrium of exofacial raft cholesterol and cytofacial non-raft cholesterol may substantially change the accessibility of membrane cholesterol pools to raft-associated proteins like secretases or APP, which might secondarily shift cellular metabolism towards the non-amyloidogenic pathway (Kirsch et al. 2003). One more possible explanation for statin action could be alteration in the neurotransmitter activity. This hypothesis can be supported with earlier studies which state that lipophilic simvastatin treatment increased D1 and D2 receptor expression (Wang et al. 2005) and also attenuated cerebral vasospasm and neurological deficits in mice, possibly by eNOS upregulation (McGirt et al. 2002). Furthermore, statins have shown the potential to render cortical neurons more resistant to NMDA-induced excitotoxic death (Zacco et al. 2003). One more important possibility for effect of statins on memory could be their action on AChE activity.

Table 6 Serum cholesterol, brain total cholesterol, AChE activity and HMG CoA reductase activity in mice brain, after long-term treatment Parameter

Control

Cholesterol

Simvastatin

Pravastatin

Serum cholesterol (mg/dl) Brain cholesterol (mg/g wet tissue) Activity of brain HMG CoA R (nmoles of mevalonate /min/mg protein) Activity of brain AchE (μ moles of substrate hydrolyzed/min)

92.43±6.08 15.20±0.33 0.218±0.01 6.78±0.10

115.70±4.22** 16.77±0.24** 0.131±0.012** 7.40±0.19*

58.44±6.73* 14.03±0.17* 0.358±0.018* 5.91±0.18**

55.05±5.24** 14.97±0.31 0.258±0.023** 6.02±0.14**

Values are expressed as Mean±SEM, statistical analysis was carried out by ANOVA followed by Dunnett's test. *p<0.05, **p<0.01, versus control group

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Simvastatin treatment for seven days had shown significant decrease in brain AChE activity in male rats (Lane and Farlow 2005). Statins may also act on several different pathways involving distinct gene expression patterns. Expression levels of genes associated with apoptosis were significantly altered by simvastatin including upregulation of the major anti-apoptotic gene Bcl-2 and downregulation of pro apoptotic gene Bax. These changes by statins were associated with a stabilization of the mitochondrial membrane potential and suppression of caspase activation in presence of various stressors. These findings provide new insights into one of multiple potential mechanisms for statins protective actions in neurological diseases where increased programmed cell death has been implicated (Franke et al. 2007). Increased expression of certain genes would be beneficial to brain cell function being capable of rescuing neurons from Aβ toxicity. Statins were found to alter the expression of these genes and proves to be potentially neuroprotective (Johnson-Anuna et al. 2005). Both simvastatin and pravastatin are specific inhibitors of HMG-CoA reductase, and the comparable efficacy of these agents in improving memory suggests the effect of a drug class. One possible explanation for this effect of statins is decrease in cholesterol level. Treatment with simvastatin as well as pravastatin showed decrease in serum cholesterol level irrespective of treatment duration. Concurrently we found increase in serum cholesterol level in high cholesterol diet group. Early work indeed suggested that serum cholesterol have only little or no influence on brain cholesterol (Kabara 1973). There is no significant change in brain cholesterol level in groups treated for short-term duration with simvastatin as well as pravastatin. However, long-term treatment with simvastatin showed significant decrease in brain cholesterol level. In contrast to simvastatin, pravastatin treatment had no significant effect on whole brain cholesterol level. The reason for the different response might be the varying transportation of the two statins across blood–brain barrier and accumulation in cell compartments. It was proposed that pravastatin is unable to penetrate cell membranes but instead accumulates inside the membrane in tissue other than the liver (Koga et al. 1990; Tsuji et al. 1993). Nelson and Alkon (2005) suggested different mechanisms as a possible explanation for the effect of cholesterol on memory. Elevation in cholesterol could increase the production of neurosteroids such as dehydroepiandrosterone, δ5-androstene-3β, 17 β-diol, pregnenolone and 7-αOH-dehydroepiandrosterone. GABAA, NMDA, cholinergic and sigma opioid systems are all potential targets of neurosteroids. Neurosteroids bind with nanomolar affinity to inhibitory GABAA receptors and with sub-micromolar affinity to excitatory glutamatergic NMDA, 5-hydroxytryptamine, Glycine, sigma type 1 and nicotinic acetylcholine receptors.

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Another possibility is that the high-cholesterol diet may affect the ratio of esterified to non-esterified cholesterol in the brain. Cholesterol esterification by acyl CoA cholesteryl acyl transferase (ACAT) has been implicated in the production of β-amyloid, the toxic peptide associated with AD. Cholesteryl esters are directly correlated with β-amyloid production and pharmacological inhibitors of ACAT can decrease β-amyloid synthesis (Nelson and Alkon 2005). The rate of local synthesis is one of the most important factors regulating cholesterol levels in the brain (Dietschy and Turley 2001). The rate of brain cholesterol synthesis can be decided by brain HMG-CoA reductase activity. We found that brain HMG-CoA reductase activity was significantly upregulated in simvastatin treated group regardless to treatment duration but, treatment with pravastatin did not cause significant alteration in HMG-CoA reductase activity even after 4 months treatment. The inhibitory effect on cholesterol synthesis in the brain due to simvastatin treatment was accompanied by a compensatory increase in HMG-CoA reductase activity (Thelen et al. 2006; Franke et al. 2007). This expected difference in activities might account for differences in capacity between the two different statins used to cross the BBB. Concurrently we found that treatment with high cholesterol diet caused a significant decrease of cholesterol synthesis in the brain as judged from down regulation of brain HMG-CoA reductase activity. However, under steady-state conditions, there is a need for elimination of cholesterol from the brain to compensate for the synthesis. 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the BBB and this hydroxylation may be critical for cholesterol homeostasis in the brain. Therefore, simultaneous estimation of 24(S)-hydroxycholesterol regarded to be a better marker for cholesterol synthesis than absolute HMG-CoA reductase activity (Bjorkhelm et al. 1997). Several studies have shown that statin actions on Aβ release are likely to be mediated by β-secretase β-site amyloid-cleaving enzyme (BACE). BACE dimerization could be inhibited by statins via HMG-CoA reductasedependent isoprenoid biosynthesis (Parsons et al. 2006). Blocking the dimerization of BACE is an attractive therapeutic target, as the dimer is 30-fold more active at cleaving APP than the monomer and so inhibiting BACE dimerization would reduce Aβ production markedly. Statins, in addition to controlling the synthesis of cholesterol, controls the synthesis of non-sterol isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Although it is clear that most of the therapeutic benefit derives from lowering plasma levels of cholesterol, it has been suggested that the associated reduction in nonsterol isoprenoid synthesis may also contribute to the beneficial outcome. Isoprenylated GTPases exert many cellular effects via activation of protein kinases such as ROCK, a member of Rho subfamily. Addition of exogenous GGPP or FPP

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specifically increases the functional activity of small GTPases and facilitates a specific increase in the secretion of Aβ42. Conversely, inhibition of Rho/ROCK was coupled to a specific decrease in Aβ42 levels (Cole and Vassar 2006). As per recent reports statin-induced inhibition of the Rho/ROCK pathway facilitates dose dependent increases in APPsα secretion and decrease in Aβ secretion (Pedrini et al. 2005). This report indicates that, at least in vitro, statins may mediate nonamyloidogenic APP processing through mechanisms other than those dependent on reduction in cellular cholesterol. In contrast to beneficial effects of statins mediated through inhibition of isoprenoid synthesis, interruption in the supply of geranylgeranyl diphosphate by statin treatment leads to loss of function of the prenylated protein, which in turn disrupts behavioral learning in whole animal (Kotti et al. 2006).

Conclusions There is no direct correlation between brain cholesterol level, as well as HMG-CoA activity and memory function regulation in animals. Short term treatment with simvastatin showed significant increase in HMG-CoAR activity in spite of unaltered brain cholesterol level. This indicates that brain cholesterol level does not regulate HMG-CoAR activity and there is some other direct mechanism responsible for HMG-CoAR activity. Pravastatin failed to alter brain cholesterol level as well as brain HMG-CoAR activity. This indicates that lipophilicity of drug play a major role in the regulation brain cholesterol level and HMG-CoAR activity. There is a direct relationship between plasma cholesterol level and brain AChE level. Cholesterol induced decrease in AChE level is not directly related with an improvement in memory function. However a long standing plasma cholesterol alteration with concurrent AChE alteration might activate some other unknown neurochemical pathway which in turn is responsible for regulation of memory function.

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