In the present study we show for the first time that hypercholesterolemia decreased the number of cholinergic neurons in the nBM and the cortical acetylcholine level, which might result in a dysfunction of the cholinergic system. Furthermore, cholesterol-enriched diet reduced spatial learning and the ability to store long-term memory in an 8-am radial maze, enhanced the inflammation process, elevated cortical beta-amyloid, tau and phospho-tau 181 and increased microbleedings in the cortex.
Cholesterol has been demonstrated to affect the APP processing and linked to the pathology of AD (Raffai and Weisgraber, 2003; Simons et al., 2001; Shobab et al., 2005
). Various animal studies have been shown that hypercholesterolemia displays some characteristics of AD in vivo (Granholm et al., 2008; Refolo et al., 2000
). The current study was performed in male rats to exclude hormonal influences, because cholesterol homeostasis of females is mediated by the hypolipodemic properties of estrogens (de Marinis et al., 2008
). Our present study revealed that a cholesterol-enriched diet enhanced plasma cholesterol concentration. However, it is not clear how plasma cholesterol is able to affect brain functions, since cholesterol does not pass the BBB. However, oxysterols, oxygenated derivates of cholesterol are able to pass lipophilic membranes (Björkhem et al., 2002; Björkhem, 2002
). In fact, increased brain levels of 27-hydroxycholesterol have been measured in AD patients (Björkhem et al., 2006
). Although, not proven enhanced blood cholesterol may be metabolized to its oxysterols, which can easier enter the brain.
The 8-arm radial maze is well established to test learning and memory in a controlled environment (Jarrard et al., 1984
). Our study revealed severe spatial learning and long-term memory deficits in hypercholesterolemic rats. Spatial memory was tested in a partially baited 8-arm radial maze. Three weeks after a 5-day acquisition period, rats were again tested to assess long-term memory performance (retention). Hypercholesterolemic rats were significantly heavier in weight and to exclude decreased mobility, we monitored various parameters and found no differences in motivation and mobility between control and hypercholesterolemic rats. We conclude that a cholesterol-enriched diet results in spatial memory impairment, which is in line with previous studies (Anstey et al., 2008; Liu et al., 2008; Foster, 2006
). Indeed, rats fed with higher amount of dietary fat showed widespread cognitive deficits on various tasks of learning and memory such as, Olton's radial arm maze, a non-spatial test of conditional associative learning, the Hebb–Williams complex maze series, and a variable-interval delayed alternation test that highlighted deficits in rule-learning and specific memory function (Greenwood and Young, 2002; Molteni et al., 2002; Winocur and Greenwood, 1999
). The mechanisms of effects of dietary fat on cognitive function are not completely understood, however, it seems possible that high fat diet may cause glucose intolerance and/or insulin resistance in rat brains (Srinivasan et al., 2004; Greenwood and Young, 2002; Molteni et al., 2002; Winocur and Greenwood, 1999; Greenwood and Winocur, 2001
AD is characterized by a substantial loss of cholinergic innervation in the cerebral cortex and a significant reduction of cholinergic basal forebrain neurons can be observed (Mesulam, 2010; Mufson et al., 2003; Vogels et al., 1990
). As a result, the concentrations of acetylcholine and ChAT are markedly reduced in the cortex of AD patients (Bowen et al., 1976; Davies and Maloney, 1976
). The clinically outcome of AD is represented by progressive memory impairment and cognitive dysfunctions (McKhann et al., 1984
). Accordingly, it has been shown that the basal forebrain cholinergic complex is closely associated with learning and memory (McKinney, 2005; Winkler et al., 1995
) and systemic disturbances lead to mnemonic impairment or loss (McLin et al., 2002; Pizzo et al., 2002
). Cholinergic neurons in the nBM have been demonstrated as the most sensitive neurons to age-related neurofibrillary degeneration (Sassin et al., 2000
) and might be associated with cognitive impairments in aging and AD (Wenk, 1997
). Our present data demonstrate that a cholesterol-enriched diet decreased the number of ChAT-positive neurons in the nBM resulting in a reduction of acetylcholine in the cortex. We suggest that hypercholesterolemia leads to a disrupted cholinergic system in the basal forebrain, which may contribute to the spatial memory impairment. Cholesterol may interact with the membrane environment of cholinergic receptors (muscarinic as well as nicotinic) and may thus modulate their activity (Bohr, 2004
). It is also very likely that there are functional relationships between cholinergic receptors and lipid rafts-specialized plasmalemmal structures enriched in cholesterol (Feron and Kelly, 2001
). Indeed, high cholesterol levels reduce the activity of plasmalemmal receptors, such as certain monoaminergic receptors, e.g. beta-adrenergic receptors (Barnett et al., 1989
), which can modulate the cholinergic neurons.
Nerve growth factor (NGF) is the most prominent molecule to protect cholinergic neurons against neurodegeneration (Schliebs and Arendt, 2006; Winkler et al., 1998
). In AD brains, a loss of NGF and its receptor TrkA was identified in the basal forebrain, whereas the concentration of NGF in target regions is increased (Schindowski et al., 2008
). This alteration is caused by an impaired retrograde transport, accumulation of NGF in target regions of cholinergic neurons and a loss of NGF in the basal forebrain (Longo and Massa, 2004; Schindowski et al., 2008; Schliebs and Arendt, 2006
). Moreover, various studies exhibited that administration of exogenous NGF can rescue memory deficits (Covaceuszach et al., 2009; de Rosa et al., 2005
). In the present study, we discovered a slight but not significant increase of NGF in the cortex of cholesterol-treated animals. Thus, we suggest that a damaged cholinergic system in hypercholesterolemia enhances NGF in the cortex.
It is well established that inflammation is seen in AD brains and in hypercholesterolemic animal models (Rahman et al., 2005; Thirumangalakudi et al., 2008; Xue et al., 2007
). Our data demonstrate that a cholesterol-enriched diet induced an elevated immunohistochemical-positive staining for the microglial marker CD11b. It has been shown, that activated microglia produce and secrete various cytokines and chemokines, e.g. IL-1, MCP-1, MIP-1α, or TNFα (Akiyama et al., 2000; Lucas et al., 2006; Rogers, 2008
). The cytokine IL-1 has been linked to APP regulation and it may promote beta-amyloid production (Akiyama et al., 2000; Eikelenboom and van Gool, 2004
) possibly by affecting the acetylcholinesterase expression (Akiyama et al., 2000
) or by activation of neurodegeneration (Akiyama et al., 2000; Stuchbury and Münch, 2005
). In agreement with previous in vivo studies, we found an activation of microglial immunoreactivity (Granholm et al., 2008; Rahman et al., 2005; Thirumangalakudi et al., 2008; Xue et al., 2007
) and elevations of different inflammation markers (Rahman et al., 2005; Thirumangalakudi et al., 2008
). The mechanism of microglial activation and the role of cholesterol in the process of inflammation is not fully known. Different studies revealed that oxygenated derivates of cholesterol (24-hydroxycholesterol, 25-hydroxycholesterol or 27-hydroxycholesterol) are involved in the upregulation of a number of inflammatory markers especially 24-hydroxycholesterol (Lemaire-Ewing et al., 2005; Rosklint et al., 2002; Dugas et al., 2010; Prunet et al., 2006; Morello et al., 2009; Trousson et al., 2009; Vejux et al., 2008; Sottero et al., 2009; Joffre et al., 2007
). We have recently shown that oxysterol treatment differentially regulate cholinergic nBM neurons in organotypic brain slices (Ullrich and Humpel, 2010
). Thus, in our hypercholesterolemic rat model, we observed strong activation of several inflammation markers, which are implicated in pathophysiological alterations similar to AD and could be mediated by different cholesterol oxysterols.
Beta-amyloid depositions and neurofibrillary tangles are distinctive hallmarks of AD. A disturbed cholesterol homeostasis within lipid rafts might influence APP processing, which result in increased beta-aymloid(1–42) production and deposition (Ghribi, 2008; Simons et al., 2001
). Whereas, little is known about the function of cholesterol in the formation of neurofibrillary tangles. Our data reveal that hypercholesterolemia enhanced APP and beta-amyloid(1–42) levels, as well as tau and phospho-tau 181 levels in the cortex. This was also confirmed by descriptive immunohistochemical analysis showing enhanced beta-amyloid-like immunoreactivity in the cortex of cholesterol-fed rats. For quantitative determination of tau and phospho-tau 181 we used a human specific ELISA assay, which has not been tested for rats. However, due to high homology between human and rat tau, we think that rat tau can be detected. Our findings are in line with previous studies showing elevated concentrations of beta-amyloid(1–42) (Sharma et al., 2008; Prasanthi et al., 2008; Ghribi et al., 2006; Refolo et al., 2000
) and an enhanced level of hyperphosphorylated tau (Ghribi et al., 2006
) after cholesterol-enriched diet. Various studies exhibited that 27-hydroxycholesterol supported beta-amyloid aggregation in the brain (Sharma et al., 2008; Prasanthi et al., 2009
). In addition, APP can be upregulated due to NGF accumulation in the cortex of AD patients (Schindowski et al., 2008
). Ghribi and colleagues (2006)
suggested that increased beta-amyloid trigger phosphorylation of tau by activation of extracellular signal-regulated protein kinase (ERK) and Nicholson and Ferreira (2009)
described an association between membrane cholesterol and beta-amyloid-induced tau toxicity in AD. Thus, we suggest that a cholesterol-enriched diet affected the cleavage of APP, which results in the production of beta-amyloid(1–42) and that hypercholesterolemia upregulates tau and phospho-tau 181 in the cortex. However, we could not observe beta-amyloid plaque deposition and neurofibrillary tangles in hypercholesterolemic rats. This findings may either need longer times of cholesterol diet or the combination with other parameters, e.g. low pH, apolipoprotein E or metals (Marksteiner and Humpel, 2008
It is well established that vascular risk factors and neurovascular dysfunction play integral roles in the pathogenesis of AD (Bell and Zlokovic, 2009; Dickstein et al., 2010; Iadecola, 2004; Stolp and Dziegielewska, 2009
). Furthermore, AD is often associated with cerebral hypoperfusion, which may lead to cortical microinfarcts and microbleedings (Suter et al., 2002; Miklossy, 2003
). It has been shown that hypercholesterolemia affects the BBB integrity and leads to increase in IgG extravasation in cholesterol-fed rabbits (Chen et al., 2008; Sparks et al., 2000
). Nevertheless, recent studies have shown that normal rat brains contain low IgG, but the levels dramatically increase after head injury (Aihara et al., 1994; Hazama et al., 2005
). In agreement, we exhibited an increased anti-rat IgG-positive immunoreactivity in the cortex of cholesterol-treated animals compared to controls indicating microbleedings and microinfarcts. In contrast, RECA-1-positive immunohistochemistry, a well-known marker for capillaries (Moser et al., 2003
) demonstrated no changes in the vascular structure of controls and hypercholesterolemic rats. However, the immunohistochemical staining against rat IgG or RECA-1 does not demonstrate changes to small molecules of the vascular system. Consequently, further studies are necessary to evaluate microchanges and the integrity of the BBB. Furthermore, it might be possible that elevated MMP-2 levels in our cholesterol-treated rats contribute to the disruption of the basal lamina and tight junctions of the BBB (Candelario-Jalil et al., 2009
). Finally, a dysfunctional transport across the BBB may also affect efflux of metabolic waste or influx of energy substrates. An example of diminished transport could be insulin, which has an important role in CNS physiology like modulation of glucose utilization in specific brain regions or modulation of synaptic levels of neurotransmitters (Biessels et al., 2004; Hoyer, 2004
). Thus, a decreased insulin transport might diminish many beneficial effects of insulin in the brain. Taken together we suggest that a hypercholesterolemic diet induces small microbleedings in the cortex associated with microglial activation and dysfunctional metabolic changes of the BBB similar to AD pathology.
Taken together, our results suggest that hypercholesterolemia causes a dysfunction of the cholinergic system, cognitive deficits, inflammation, beta-amyloid and tau-pathology and microbleedings. Thus, hypercholesterolemic rats resemble some AD-like pathologies and demonstrate for the first time an impaired cholinergic system due to high cholesterol diet.