Astrocytosis, microgliosis, metallothionein-I-II and amyloid ... - IOS Press

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Astrocytosis, microgliosis, metallothionein-I-II and amyloid expression in high cholesterol-fed rabbits Paolo Zattaa,∗ , Pamela Zambenedetti b , Maria Pia Stellac and Federico Licastro d a

CNR Center on Metalloproteins, Department of Biology, University of Padova, Italy b Division of Anatomopathology, General Hospital, Dolo, Venice, Italy c Sant’Antonio Hospital, Padova, Italy d Department of Biomedical Sciences, School of Medicine, University of Bologna, Italy Cholesterol is considered a risk factor in vascular dementia as well as in Alzheimer’s disease. Several biochemical, epidemiological and genetic aspects established a correlation between cholesterol concentration and Alzheimer’s disease. Microglia activation, astrocytosis with metallothionein-I-II overexpression, amyloid β intraneuronal accumulation and a rare formation of amyloid β extracellular positive deposits were the major immunohistochemical features observed in the brain of high cholesterol-fed animals. The relevance on the cholesterol metabolism in Alzheimer’s disease pathogenesis is also discussed. Keywords: Aβ metabolism, Astrocyte activation, microgliosis, Cholesterol-fed rabbit, Metallothionein

1. Introduction The brain is rich in cholesterol, where is synthesized in situ [20]. However, cholesterol dynamics in the brain (e.g., transport, metabolism etc.) remain to be defined. Studies have demonstrated that cholesterol synthesis is high in the fetal brain and decreases as the animal matures [11,51]. Some recent data suggest the cholesterol ∗ Corresponding

author: Paolo Zatta, CNR Center on Metalloproteins, Metals and the Brain Laboratory, Dept. of Biology, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy. Tel.: +39 49 827 6331; Fax: +39 49 827 6330; E-mail: zatta@cribi1.bio.unipd.it. Journal of Alzheimer’s Disease 4 (2002) 1–9 ISSN 1387-2877 / $8.00  2002, IOS Press. All rights reserved

content is slightly but significantly increased in the grey matter of the frontal cortex of AD patients [44]. Diversely, other studies concluded that the brain cholesterol content may be lower in AD patients than in agedmatched controls [27]. Changes in intracellular cholesterol levels might contribute to neuronal degeneration associated with AD by decreasing the production of soluble amyloid [3] and increasing the insoluble fragment. An association between cholesterol and AD also comes from epidemiological data indicating a relationship between the apolipoprotein E (ApoE) ε4 allele and the occurence of AD [48,53]. ApoE is a genetically polymorphic plasma protein with regulatory role in lipid transport and cholesterol homeostasis. In humans, ApoE occurs as three major isoforms named E2, E3, E4. Genetic evidences demonstrates an association of ApoE ε4 allele in AD, as well as with other pathological events such as cholelithiasis, lipid dismetabolism and atherosclerosis [7,47,53]. The role of ApoE in central nervous system (CNS) is particularly important in relation to the function of cholinergic system, which relies to a certain extent on the integrity of phospholipid homeostasis in neurons and it is substantially altered in AD [32]. High plasma cholesterol levels have been found in ApoEε4 homozygous demented subjects [9], and high concentrations of cholesterol have been shown to modify the function of certain membrane proteins from cultured HEK293 cells [3]. Interesting observations also derive from animal models. For instance, amyloid β intracellular deposition in brains from rabbits under a hypercholesterol diet has been reported [42]. However, only 1 out of 13 animals showed extracellular deposition of amyloid β [41,46]. These findings suggested that elevated cholesterol alone could not be the cause of extracellular amyloid β deposition, and that other yet-to-be-defined factors might also mediate plaque formation [43,46]. Nevertheless, the increased concentration of amyloid might induce microglial cell migration and activation.

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P. Zatta et al. / Astrocytosis, microgliosis, metallothionein-I-II and amyloid expression

In fact, activated astroglia and microglia surround the amyloid deposits and neuritic plaques in the brain of AD patients [54]. Metallothioneins (MT) are a large family of intracellular, low molecular weight, cystein-rich proteins [26]. In humans, MT are encoded by a complex multigene family located on chromosome 16, which includes two functional MT-I genes and one functional MT-II gene [36]. The physiological function of MT are not yet fully established. In the brain, MT are expressed in three forms, MT-I, MT-II and MT-III. MT-I and II were identified prevalently in astrocytes, but they are also detecable in the pia mater, ependymal cells, glial processes and fibrous astrocytes from the white matter, while they are absent in neurons, oligodendroglia and microglia [17]. MT-III, appears to be expressed mostly in neurons [17]. A general consensus exists on the role of MT in the copper and zinc homeostasis as well as in their protective role against cytotoxicity induced by metal ions and by reactive oxygen species [30]. Our recent findings showed that the expression of MT-I-II is significantly higher in the cortex, cerebral white matter and cerebellum of AD than control brains [58]. AD brain sections revealed high expression of MT-I-II in astrocytes and capillaries, as well as in the granular, but not in the molecular layer of cerebellum. In this paper we report that high cholesterol-fed rabbits show an elevated immunoreactivity for MT-I-II particularly in astrocytes of the white matter and a low immunopositivity also in the grey matter. On the contrary, brains from controls were negative for MT-I-II. Moreover, intraneuronal and rare extraneuronal deposits of amyloid β, microgliosis and activated astrocytes immunoreactive for MT-I-II in high cholesterol-fed rabbits were also observed.

2. Material and methods 2.1. Immunocytochemistry Thirteen male New Zealand White rabbits (NZW) were treated for four months with a 2% cholesterol supplementation (Morini, Bologna, Italy) and drinking water, ad libitum; 5 male NZW controls were fed a normal diet. The brain was quickly removed after animal sacrifice and immediately fixed in 10% formalin buffered with Tris-HCl µ = 0.1, pH 7.5. Paraffin sections with a thickness of 6 µm were prepared for immunohistochemical studies. Immunohistochemistry

was carried out as follows. Dewaxed sections were incubated in 3% H 2 O2 diluted in PBS for 10 min; after washing, sections were treated with normal goat serum for 30 min, then incubated overnight at 4 ◦ C with 1:50 dilution of anti-metallothionein-I+II antibody (antiMTs; DAKO, Milan, Italy), washed twice with PBS, then reacted for 30 min with biotinylated goat antimouse immunoglobulin G, rinsed three times, and processed with streptoavidin-peroxidase complex (ABC complex; DAKO). Immunostain was developed by addition of 3-3’-diaminobenzidine and substrate chromogen solution, then counterstained with hematoxylin. Activated or reactive astrocytes were detected by using either an anti-GFAP polyclonal antibody (DAKO, Milan, Italy) or an anti-S-100 polyclonal antibody (DAKO, Milan, Italy). Amyloid deposits were stained by polyclonal rabbit antibody specific for amyloid βAlzheimer (Roche, Milan, Italy). The absence of cross reactivity was confirmed using rabbit anti-CD3 polyclonal antibody. All antibodies utilized in our study are reported in Table 1. 2.2. Silver impregnation staining was carried out following the Bielschowsky method as modified by Yamamoto and Hirano [57] 2.3. Analyses Serum levels of cholesterol, triglycerides, ApoA and Apo B were determined using specific kits purchased from Dade Behring, Milan, Italy. HDL-C type test, an in vitro assay, for the quantitative determination of high density lipoprotein cholesterol in serum, was carried out using a kit from Wako Chemicals GmbH, Neuss, Germany. 2.4. Statistical evaluation Comparison between groups was performed according the two tailed Student-t test. Correlations between experimental variables were identified by linear regression analysis and the correlation coefficients were calculated. 2.5. Photomicrography The processed sections were photographed with a Nikon F-601 camera coupled to a Leitz Diaplan microscope, using Kodak Elite II (ISO 100/21 0).


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Table 1 Characteristics of antibodies and lectin utilized in the immunohistochemical study Antibody MT-I-II S-100 Amyloid β Ferritin GFAP MCA∗ Presenilin-1 Ubiquitin Synaptobrevin Synaptophysin Neurofilament-H Neurofilaments Phosphorylated ∗ MCA

Producer DAKO, Milan, Italy DAKO, Milan, Italy Roche, Milan, Italy DAKO, Milan, Italy DAKO,Milan, Italy Vector, DBA, Segrate, Italy Calbiochem, Milan, Italy DAKO, Milan, Italy Oncogene, Cambridge, MA, USA Oncogene, Cambridge, MA, USA Affinity, Mamhead Castle, UK Affinity, Mamhead Castle, UK

Source Mouse Rabbit Rabbit Rabbit Rabbit – Rabbit Rabbit Mouse Mouse Mouse Mouse

Dilution 1:50 1:200 1:10 1:100 1:100 1:10 1:25 1:100 1:50 1:100 1:100 1:100

Pre-treatment None and trypsin Trypsin Trypsin None Trypsin None None Heating in 10 mM citrate buffer None None None None

= Momordica caranthia agglutinin.

3. Results 3.1. Lipid profile The lipid profiles of the two groups of rabbits under different diets are reported in Tables 2A and 2B. Circulating levels of ApoA, ApoB, cholesterol, cholesterolHDL and cholesterol-LDL were significantly increased in high-cholesterol fed animals. The positivity of immunoreactivity for MT-I-II was transformed in numeric indexes (+ = 1, ++ = 2, + + + = 3) and then correlated with serum levels of lipids and lipoproteins. Serum levels of lipids and lipoprotein from high colesterol-fed animals positively correlated with immunoreactivity for MT-I-II in the white matter. In particular, serum levels of ApoA, cholesterol, cholesterolHDL and LDL significantly correlated with the levels of the brain MT-I-II. On the contrary, no correlation between serum triglycerides and brain MT-I-II was observed. 3.2. Histopathological findings Figure 1 shows the Bielschowsky silver impregnation staining pattern of rabbit brain cortex sections. In the controls neurofilaments are the only elements stained (Fig. 1(A)). In contrast, neurofilaments, neurons and glial cells in the cortex of high cholesterol-fed rabbits were positively stained (Fig. 1(B)), in neurons chromatin staining being particularly evident. Astrocytes were first identified by immunohistochemistry using anti-GFAP and anti-S100 antibodies. Increased MT-I-II immunoreactivity was also observed in astrocytes from high cholesterol-fed animals. In fact, in these animals MT-I-II was weakly increased in the astrocytes of grey matter, but yielded a strong strain-

ing pattern in the white matter. Controls were negative for MT-I-II immunoreactivity in the grey matter and weakly positive or negative in the white matter. Figure 2 shows results of the immunohistochemistry carried out with anti-metallothionein-I-II; controls appear to be negative (Fig. 2(A)), while the cortex of high cholesterol-fed animals contained both immunopositive astrocytes, and endothelial cells (Fig. 2(B)). Activated microglial cells were identified by anti-ferritin antibody and by Momordica caranthia lectin [58]. Sections of the brain cortex from high cholesterolfed animals were stained by anti-amyloid β antibodies. High cholesterol-fed animals showed a strong amyloid β positivity in about 10% of neurons (Fig. 3(C)), and rare diffuse type plaques were observed (2–3 per section) only in two animals out of 13 under high cholesterol diet, but in none of the controls (Figs 3(A), (B) and (D)). Table 3 summarizes most relevant histopathological findings. Among the high cholesterol-fed animals, amyloid β positivity was detected in neurons of 2 animals, in the meninges of 4 animals and in the capillaries of all 13 animals. Immature plaques were present only in 2 out of 13 animals. All controls tested were negative for amyloid β immunoreactivity. No difference regarding immunoreactivity for other antibodies listed in Table 1 between the two animal cohorts was observed.

4. Discussion During aging a marked increase in the ratio of cholesterol/phospholipids in the cell membranes from various tissues has been observed, with special regard to the brain [23,35,50]. A link between AD and cholesterol metabolism may derive from the finding that subjects

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P. Zatta et al. / Astrocytosis, microgliosis, metallothionein-I-II and amyloid expression Table 2A Lipid profile in peripheral blood from rabbits fed high-cholesterol or normal diet

ApoA (mg/dl) ApoB (mg/dl) Cholesterol (mg/dl) Cholesterol-HDL Triglycerides (mg/dl) Cholesterol-LDL (mg/dl)

High cholesterol diet (n = 13) 25 ± 16 138 ± 113 741 ± 393 299 ± 181 102 ± 128 500 ± 216

Normal diet (n = 5) 4.0 ± 0.2 28 ± 0.2 33 ± 8 18 ± 5 72 ± 12 5±2

p 0.0008 0.004 0.0003 0.0001 n.s. 0.00003

Data are presented as mean ± S.D.; statistical comparisons (p) have been performed according the two tailed Student-t test.: n.s. = not statistically significant.

with ApoE ε4 allele have a modified risk for late-onset AD [25]. However, no direct evidence has been reported so far linking cholesterol metabolism and the etiopathogenesis of AD. The amyloid β protein precursor (AβPP), and ApoE molecule appear genetically linked to AD, since APP mutations are found in familial AD and the ApoE ε4 allele is associated with both familial or sporadic AD [32]. During intracellular transport AβPP undergoes several proteolytic cleavages which lead to the release of an amyloidogenic fragment or a non-amyloidogenic secreted form [38]. Cholesterol decreases the secretion of the soluble fraction of amyloid β protein precursor (sAβPP) by interfering with maturation as well as glycosylation processes [15]. In addition, by decreasing membrane fluidity, cholesterol may lower sAβPP production by impeding the interaction of the precursor substrate with proteases. Furthermore, amyloid β decreases cholesterol esterification and changes the distribution of free cholesterol in neurons [25]. For instance, incubation of COS cells with increasing concentrations of nonesterified cholesterol caused a dose-dependent inhibition of sAPP and cell cholesterol mass inversely correlated with sAβPP release [33]. In mice, it has been reported that sAβPP processing derivatives and amyloid β could be modulated in the brain by diet and cholesterol modulated AβPP processing in vivo [18]. Thus, alterations of cellular cholesterol concentrations in AD might contribute to neuronal degeneration by decreasing sAβPP production and favoring the formation of amyloidogenic AβPP devivatives [3]. It has been demonstrated that the formation of amyloid β in hippocampal neurons is completely inhibited by reducing cellular cholesterol levels using drugs such as lovastatin and methyl-β-cyclodextrin [38]. It is noteworthy that the binding affinity of cholesterol for amyloid polymers is markedly higher with respect to other lipids like phosphatidylcholine and saturated fatty acids [2].

Table 2B Regression analysis between lipid profile data and metallothioneinI-II immunoreactivity in the brain white matter from 18 rabbits fed high-cholesterol diet or normal diet ApoA/MT ApoB/MT Cholesterol/MT Cholesterol-HDL/MT Tryglicerides/MT Cholesterol-LDL/MT

Correlation coefficient 0.63 0.26 0.49 0.6 0.003 0.61

p 0.007 n.s. 0.04 0.008 n.s. 0.007

n.s.= not statistically significant.

The present study reveales that high cholesterol-fed rabbits present some neuropathological features similar to those observed in AD brains. NZW fed a 2% cholesterol diet exhibited activated microglial cells and astrocytes, and amyloid β positive staining of some neurons. In addition, we observed a rare formation of senile plaque-like structures in two out 13 rabbits. It is noteworthy that abnormal activation of microglial cells may play a role in the etiopathogenesis of AD. In fact, the senile plaques of AD brains are surrounded by activated microglial cells and astrocytes [54]. Recently, using a transgenic mouse model for AD amyloidosis, it has been demonstrated that high cholesterol diet significantly increased amyloid β load in the brain. These data, as well as our results herein reported, support the notion that high dietary cholesterol by increasing amyloid β accumulation may accelerate AD manifestation. It is well known that 3-hydroxyglutaryl coenzyme A reductase inhibitors, known as statins, by reducing cholesterol levels, prevents coronary heart disease. This compound may have additional neuroprotective properties such as, antioxidant and anti-inflammatory effects that might have potential therapeutic utilization in neurodegeneration associated with AD [8]. Recently, some authors reported that guinea pigs treated with high doses of statins showed a strong and re-


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Fig. 1. Silverstained sections of rabbit cerebral cortex. While neurofilaments are the only structures stained in the control (A), both neurofilaments, neurons, and glial cells are stained in sections from the treated rabbit (B), with staining of chromatin in neurons particularly evident (x 40).

Fig. 2. Immunohistochemistry of rabbit cerebral cortex carried out using anti-metallothionein-I-II antibody. Control sections are completely negative (A). In contrast, sections from rabbits subjected to a high-cholesterol diet (B) show positive staining in both endothelial cells and astrocytes (x 40).

versible reduction of cerebral amyloid β peptide 1−42 and amyloid β peptide 1−40 levels in the CSF and brain homogenate [12]. Apparently, statins do not reduced levels of total brain cholesterol, but the availability of the cholesterol precursor lathosterol, indicating that the treatment interferes with the de novo brain cholesterol synthesis [29]. In spite of some encouraging preliminary results, the association of statin with AD is not definely proved [56]. However, studies in a transgenic animal model of AD have demonstrated that administration of non-steroidal anti-inflammatory drugs like ibuprofen or indomethacin can significantly delay amyloid deposition [24,31]. In conclusion, the link between amyloid and AD as a primary or secondary etiopathogenic factor is still matter of debate indicat-

ing that the amyloid hypothesis needs further studies to determine whether amyloid deposition is a primary factor in AD. Microglial cells produce IL-1 as well as other cytokines which drive highly activated microglial cells into plaques, thus creating a vicious circle. In addition, IL-1 stimulates other cells to produce amyloid β and reactive oxygen species [5] which augment amyloid clumping, resulting in activation of other microglial cells and consequently further amyloid formation and deposition [45]. Streit and Sparks [46] observed elevated microglial cell activation in the brains of humans with heart disease as well as in the brains of hypercholesterolemic rabbits. Microgliosis, accompanied by the accumulation of amyloid β immunoreactivity, was

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Fig. 3. Immunocytochemistry of rabbit cerebral cortex carried out using anti-amyloid β antibody. While the section from the control rabbit scores negative (A), sections from rabbits subjected to a high-cholesterol diet (B) show strong positive staining neurons (B, C); some animals also show diffusely staining plaques (B, D). (x 40).

also observed in cholesterol-fed rabbits [43]. This accumulation persisted after the animals were withdrawn from the high cholestrol diet and placed on a normal diet. In this paper it is reported that Apo-A and Apo-B increased 6 and 5 times respectively in high cholesterolfed rabbits. On the other hand, cholesterol-LDL levels were 100 time higher than those from control animals (Table 2 and 2A). Cytokines increase cholesterol synthesis and lipoprotein secretion resulting in elevated cholesterolemia [14]. Cholesterol 7-α-hydroxylase (CAH) is the rate-limiting enzyme in the conversion of cholesterol to bile acids [1]. In hamster, IL-1 administration produced a marked decrease in the level of CAH mRNA. In animals fed a cholesterol-enriched diet, a marked decrease of CAH mRNA was also observed [13]. In vertebrates, ApoA-I is the major pro-

tein of high-density lipoproteins and may play an important role in regulating tissue cholesterol content by reversing the cholesterol transport pathway. Brain cells are able to produce cytokine, acute-phase proteins, complement proteins, prostaglandins and free radicals. In AD activated microglia and astrocytes surround amyloid deposits and neuronal axons with neurofibrillary tangles. In a previous paper we have demonstrated that MT-I-II immunoreactivity was increased in the brain from AD patients [58]. Herein it has been shown that MT-I-II immunoreactivity is high in astrocytes from rabbits fed a high cholesterol diet, along with the activation of astrocytes and a high blood cholesterol-LDL level. A number of reports demonstrated that the expression of MT-I-II was upregulated by inflammatory factors [10,37] and by stress [19]. Thus, MT-I-II expression in CNS may reflect the as-


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Table 3 Expression of amyloid β, metallothionein-I-II and ferritin in the NZW brains after treatment with 2% cholesterol diets and the controls Animal

βA4

1 2 3 4 5 6 7 8 9 10 11 12 13 C1 C2 C3 C4 C5

N, V V, M V, M, PL V, PL V, PL, P V, PL V, PL, N V, PL V V, PL + Lipids V V, PL, M V, M − − − − −

MT-I-II Grey matter + + +/− − − − +/− + + ++ +/− ++ − − − − − −

MT-I-II White matter +++ +++ ++ ++ ++ ++ ++ +++ ++ ++ ++ +++ + − +/− − +/− −

Ferritin V, MGA “ “ “ V V, MGR “ “ V V, MGR V − − − − − − −

+ = 3–6; ++ = 7–9; + + + > 10 MT-I-II positive astrocytes x mm2 . Animals treated with 2% cholesterol diet (1–13); controls (C1–C5). N = Neurons; V = Vessels; M = Meninges; MGA = Microglial cells activated; MGR = Microglial cells resting; PL = Plexus; P = Plaques; C = Control.

troglia response associated with brain damage. In fact, traumatic or chemical injury of the CNS elicits an activation of glial cells mediated by the release of proinflammatory cytokines. IL-1 is a potent stimulator of MT-I-II induction in astrocytes. The activation of astrocytes by production of pro-inflammatory cytokines might increase the expression of MT, possibly to protect against the augmented production of oxygen radicals induced by activated microglia. As shown here, brains from high cholesterol-fed animals indeed showed increased immune reactivity for markers of microglia activation. In AD, as well as in other neurodegenerative diseases, phospholipid and neutral lipid composition is greatly modified [40]. The lipid and cholesterol composition of white matter and myelin from semioval centers of AD patients has been observed to be reduced by 70% [52]. Furthermore, among demented patients the highest plasma cholesterol levels were found in the ApoE(4 homozygous AD [9]. Increased dietary cholesterol leads to a significant reduction in brain levels of secreted APP derivatives. This alteration was observed to be negatively correlated with serum cholesterol and brain ApoE thus suggesting that AβPP processing derivatives and amyloid β can be modulated in the brain by diet. Cholesterol indeed plays a role in the modulation of AβPP processing in

vivo [18]. According to some authors [3], cholesterol, by decreasing membrane fluidity, may reduce sAβPP production by impeding the interaction of the precursor substrate with proteases, and decreased sAβPP may contribute to neuronal degeneration. It has been also reported that cholesterol/phospholipid ratio was significantly lower in the platelets of patients with AD [6]. In particular, plasma from AD patients presents a decrease in cholesterol esterification; this reduction can also be induced by amyloid β peptides with an inhibition rate of 40–50% [22]. Small angle X-ray diffraction analysis of AD lipid membranes reconstituted from cortical grey matter showed a significant (30%) reduction in lipid bilayer width and a significant decrease in the unesterified cholesterol/phospholipid molar ratio [28]. In contrast, X-ray diffraction analysis of cholesterol-enriched AD samples demonstrated a virtual restoration of a normal membrane bilayer width. These findings suggest that cholesterol may affect the AD lipid membrane structure with possible deleterious consequence for the catabolism of membrane-bound proteins, such as AβPP [28]. Furthermore, biochemical analysis of thrombocyte membrane preparations from AD patients showed a reduced cholesterol content with respect to age-matched controls; such a reduction was not observed in vascular dementia [4]. Other authors reported

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that cholesterol esterification was 16% lower in AD patients, with correlation analysis of lecithin/cholesterol acyltransferase activity and plasma lipids revealing additional differences between AD and control subjects [21]. Our data support the notion that cholesterol may be implicated in amyloid formation and deposition, as well as astroglia activation, as shown by the high expression of the heat shock proteins, MT-I and MT-II.

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Acknowledgment

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Authors are grateful to Ms P. Cecchetto, Ms V. Viale, Ms C. Renest, Ms P. Boato, Ms S. Cassandro and Mr G. Lamon for their technical assistance and Dr. J.L. Davis for the manuscript revision.

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