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. 2017 Dec 20;7(1):17922.
doi: 10.1038/s41598-017-17911-z.

Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

Rajib Paul  1   2 , Anupom Borah  3
Affiliations

Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

Rajib Paul et al. Sci Rep. .

Abstract

There exists an intricate relationship between hypercholesterolemia (elevated plasma cholesterol) and brain functions. The present study aims to understand the impact of hypercholesterolemia on pathological consequences in mouse brain. A chronic mouse model of hypercholesterolemia was induced by giving high-cholesterol diet for 12 weeks. The hypercholesterolemic mice developed cognitive impairment as evident from object recognition memory test. Cholesterol accumulation was observed in four discrete brain regions, such as cortex, striatum, hippocampus and substantia nigra along with significantly damaged blood-brain barrier by hypercholesterolemia. The crucial finding is the loss of acetylcholinesterase activity with mitochondrial dysfunction globally in the brain of hypercholesterolemic mice, which is related to the levels of cholesterol. Moreover, the levels of hydroxyl radical were elevated in the regions of brain where the activity of mitochondrial complexes was found to be reduced. Intriguingly, elevations of inflammatory stress markers in the cholesterol-rich brain regions were observed. As cognitive impairment, diminished brain acetylcholinesterase activity, mitochondrial dysfunctions, and inflammation are the prima facie pathologies of neurodegenerative diseases, the findings impose hypercholesterolemia as potential risk factor towards brain dysfunction.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Timelines of the experiment. Abbreviations: OLM, object location memory; ORM, object recognition memory; BBB, blood-brain barrier; AChE, Acetylcholinesterase; •OH, hydroxyl radical; GFAF-IR; Glial fibrillary acidic protein-Immunoreactivity; NOS, nitric oxide synthase.
Figure 2
Figure 2
Hypercholesterolemia on cognitive functions in mice. (A) Short-term Object Location Memory (OLM; 90 min) and (B) Long-term Object Recognition Memory (ORM; 24 h). DI was calculated as: [(time exploring the novel object − time exploring the familiar)/(time exploring novel + familiar) × 100]. The results are mean ± S.E.M. **P ≤ 0.01 as compared to control. Data were analyzed using an unpaired Student’s t-test.
Figure 3
Figure 3
High-cholesterol diet causes hypercholesterolemia, increases cholesterol level in brain and disrupts blood-brain barrier (BBB). Control (CON) mice received normal diet and the HCD group received high-cholesterol diet (at 5% w/w mixed with normal diet) for 12 weeks and sacrificed on the last day. (A) Cholesterol level in blood serum (n = 8). (B) Cholesterol level in brain. Brain cholesterol level was assayed histologically in paraformaldehyde fixed tissue sections following Schultz’s method. Photographs are representative sections showing the cortex (CX), striatum (NCP), hippocampus (HP) and substantia nigra (SN) from control and HCD mice. The blue or greenish-blue colour denotes cholesterol. The dark circles denote air bubbles formed as a result of acid (sulphuric acid: acetic acid) reaction. Photographs were taken at 4× magnification. (C) Estimation of brain cholesterol level. Cholesterol level was estimated from tissue homogenates of CX, NCP, HP and SN regions of brain by using kit (n = 6). (D) BBB disruption. Evans Blue dye (EBD) extraversion in brain tissues were analysed for possible disruption of BBB using a Spectrophotometric method (n = 4). The results are mean ± S.E.M. *P ≤ 0.05 or **P ≤ 0.01 as compared to control. Data of cholesterol level in serum (A) and brain EBD were analyzed using an unpaired Student’s t-test (D), and brain cholesterol levels were analyzed using a two-way repeated measure ANOVA (C).
Figure 4
Figure 4
Hypercholesterolemia reduces acetylcholinesterase (AChE) activity in brain. Animals were sacrificed and perfused with 10% glycerol on the last day of 12 weeks on normal diet (control; CON/CS) or high-cholesterol diet (HCD). 20 µm coronal serial sections of different brain regions were processed for AChE histoenzymology. Photographs are representative sections of AChE staining showing the cortex (CX; A1 and A2), striatum (NCP; B1 and B2), hippocampus (HP; C1 and C2) and substantia nigra (SN; D1 and D2) of CON and HCD mice. Photographs were taken at 4× magnification. Activity of AChE in CX (A3), NCP (B3), HP (C3) and SN (D3) were quantified using Ellman’s method. Intended brain regions were dissected out and processed for the estimation of AChE activity by taking absorbency at 412 nm (n = 6). AChE activity decreased significantly in all the regions. Data represent AChE activity in mu/mg protein; and mean ± SEM. *P ≤ 0.05 and **P ≤ 0.01 as compared to CON of the respective regions of brain. P-values were calculated using a two-way repeated measure ANOVA.
Figure 5
Figure 5
Hypercholesterolemia (A) diminishes mitochondrial complex-I activity and (B) elevates hydroxyl radical (•OH) level in brain. Mice were subjected to standard diet (control; CON) or high cholesterol diet (HCD) daily for 12 weeks. Animals were sacrificed by decapitation on the last day of treatment, different regions of brain, such as cortex (CX), striatum (NCP), hippocampus (HP) and substantia nigra (SN) were quickly dissected out and processed for specific activity assay of mitochondrial complex-I using NADH as a substrate and represented as nmol of NADH oxidized/min/mg protein (n = 6). For estimating the levels of •OH, animals were sacrificed 2 h post administration of salicylic acid (100 mg/kg; i.p.) on the last day of treatment. 2,3- and 2,5-dihydroxy benzoic acid (DHBA; •OH adducts of salicylate) formed were measured from tissue homogenates of intended brain regions using a sensitive HPLC-ECD method to measure the level of total DHBA. Data are expressed as pmol of DHBA per mg tissue. Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON (n = 6). A two-way repeated measures ANOVA was used to calculate P-values.
Figure 6
Figure 6
Hypercholesterolemia diminishes mitochondrial complex-II activity in brain. Representative photographs of histoenzymological staining of mitochondrial complex-II activity in cortex (CX; A1 and A2), striatum (NCP; B1and B2), hippocampus (HP; C1 and C2) and substantia nigra (SN; D1 and D2) regions of brain of control (CON/CS) and high-cholesterol diet (HCD) groups of mice. Photographs were taken at 4× magnification. Densitometric analysis of the photographs of complex-II staining (A3,B3,C3,D3). Photographs of four serial sections of CX (A3), NCP (B3), HP (C3) and SN (D3) of each group (n = 4) were analyzed using ImageJ software for determination of optical density. Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON. P-values were calculated using an unpaired Student’s t-test (two-tailed).
Figure 7
Figure 7
Hypercholesterolemia diminishes mitochondrial complex-III activity in brain. Representative photographs of histoenzymological staining of complex-III activity in cortex (CX; A1 and A2), striatum (NCP; B1 and B2), hippocampus (HP; C1 and C2) and substantia nigra (SN; D1 and D2) of control (CON/CS) and high-cholesterol diet (HCD) groups of mice. Photographs were taken at 4× magnification. Densitometric analysis of the photographs of complex-III staining (A3,B3,C3,D3). Optical density was analyzed from four serial sections of CX (A3), NCP (B3), HP (C3) and SN (D3) regions of brain of each group (n = 4). Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON. An unpaired Student’s t-test (two-tailed) was used to calculate P-values.
Figure 8
Figure 8
Hypercholesterolemia causes inflammation in glia. Animals were sacrificed and perfused with 4% PFA following 12 weeks (84th day) on normal diet (control; CON) or high-cholesterol diet (HCD). The coronal sections passing through CX (A,B), NCP (C,D), HP CA1 (E1,F1), HP CA3 (E2,F2), HP DG (E3,F3) and SN (G–H) were processed for Glial fibrillary acidic protein (GFAP)-immunoreactivity. GFAP-reactivity was increased in all the brain regions of hypercholesterolemic mice (B,B1,D,D1,F,F1–F3,H,H1) compared to the corresponding brain regions of control mice (A,A1,C,C1,E,E1–E3,G,G1), which signifies astrocytosis due to inflammation. Photographs were taken at 4× and 20× magnification. CX = cortex; NCP = striatum; HP = hippocampus; SN = substantia nigra; CA = Cornus ammonis; DG = Dentate Gyrus.
Figure 9
Figure 9
Hypercholesterolemia causes inflammation in neurons. Nitric oxide synthase (NOS) activity in cortex (A,B,I,J), striatum (C,D,K,L), hippocampus (E,F,M,N) and substantia nigra (G,H,O,P) regions of brain was analyzed by histoenzymology for possible inflammatory stress in neurons. NOS-active neurons were more in (B,J) cortex, (D,L) striatum, (F,N) hippocampus and (H,P) substantia nigra regions of hypercholesterolemic mice, compared to the corresponding brain regions of control mice (A,C,E,G). Arrows point to NOS-active neurons.
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