Specific loss of brain ABCA1 increases brain cholesterol uptake and influences neuronal structure and function

Abstract

The expression of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) in the brain and its role in the lipidation of apolipoproteins indicate that ABCA1 may play a critical role in brain cholesterol metabolism. To investigate the role of ABCA1 in brain cholesterol homeostasis and trafficking, we characterized mice that specifically lacked ABCA1 in the CNS, generated using the Cre/loxP recombination system. These mice showed reduced plasma high-density lipoprotein (HDL) cholesterol levels associated with decreased brain cholesterol content and enhanced brain uptake of esterified cholesterol from plasma HDL. Increased levels of HDL receptor SR-BI in brain capillaries and apolipoprotein A-I in brain and CSF of mutant mice were evident. Cholesterol homeostasis changes were mirrored by disturbances in motor activity and sensorimotor function. Changes in synaptic ultrastructure including reduced synapse and synaptic vesicle numbers were observed. These data show that ABCA1 is a key regulator of brain cholesterol metabolism and that disturbances in cholesterol transport in the CNS are associated with structural and functional deficits in neurons. Moreover, our findings also demonstrate that specific changes in brain cholesterol metabolism can lead to alterations in cholesterol uptake from plasma to brain.

Figure 1.

Brain and plasma cholesterol levels in Abca1^−B/−B mice. A, Selective loss of ABCA1 in the brains of Abca1^−B/−B mice. Tissue lysates from different brain regions, liver, and small intestine from control (+/+) and Abca1^−B/−B mice were analyzed by immunoblotting using a polyclonal antibody for ABCA1. GAPDH was used as a loading control. Densitometry analysis of the liver and intestine blot is shown (means ± SEM, n = 4). B, Cholesterol levels in cortex and hippocampus were analyzed by gas-liquid chromatographic mass spectrometry. **p < 0.01 versus control littermates, n = 6 per group. C, Levels of total cholesterol (TC), HDL-cholesterol (HDL-C), phospholipids (PL), and triglycerides (TG) in plasma measured by enzymatic assays. *p < 0.05, versus control littermates, n = 12–20 per group. Ctx, Cortex; Hipp, hippocampus; Cb, cerebellum; BG, basal ganglia.

Figure 2.

Brain selective uptake of CE from plasma HDL. Control (n = 4) and Abca1^−B/−B (n = 5) mice were injected with HDL particles labeled with ¹²⁵I-TC in the apolipoprotein and ³H-CEt in the CE moiety, respectively, and plasma clearance and tissue uptake of ¹²⁵I-TC and ³H-CEt were measured. Data are shown as means ± SEM. A, Fraction of ³H and ¹²⁵I activity remaining in plasma at 24 h. B, Brain uptake rate, measured by brain FCR, of ³H-CEt and ¹²⁵I-TC. C, Selective brain ³H-CE uptake rate. D, Regression analysis of correlation between the fraction of ³H cpm left in plasma at 24 h and brain ³H-CE uptake (black boxes: control mice, gray triangles: Abca1^−B/−B mice). *p < 0.05, **p < 0.01 compared with control mice.

Figure 3.

Brain sitosterol levels. Sitosterol levels were analyzed by GC-MS in cortex (Ctx) and hippocampus (Hipp) of control and Abca1^−B/−B mice. Data are shown as means ± SEM. *p < 0.05, ***p < 0.001 versus control mice; n = 6 per group.

Figure 4.

Levels of SR-BI in brain capillaries from Abca1^−B/−B mice. Brain capillaries were isolated from control and Abca1^−B/−B mice. A, A representative of a Western blot for the BCEC marker PECAM-1. PECAM-1 signal was found in the isolated capillaries (lanes 5–8) in control and Abca1^−B/−B mice but not in the capillary-depleted brain tissue (lanes 1–4). GAPDH was used as loading control. B, Capillaries were immunoblotted for SR-BI and ABCA1 (n = 4 per group). Actin was used as a loading control. Graph shows means ± SEM of densitometric measurements of SR-BI and ABCA1 signal intensity. Blots are representative of several experiments. **p < 0.01 compared with control mice.

Figure 5.

ApoE and ApoA-I levels in the brain and CSF of Abca1^−B/−B mice. A, Immunoblots of brain tissue from control and Abca1^−B/−B mice using ApoE and ApoA-I specific antibodies. Tubulin was used as a loading control. Densitometry analysis demonstrated a 40% decrease in ApoE and a 250% increase in ApoA-I signal intensity in Abca1^−B/−B mice compared with control mice. Graph shows means ± SEM (*p < 0.05, **p < 0.01, n = 3 per group). B, CSF samples (3 μl) were run on a nondenaturing gel and probed for ApoE (left) and ApoA-I (right).

Figure 6.

Brain cholesterol turnover in Abca1^−B/−B mice. Brain and plasma levels of cholesterol metabolite 24S-OH cholesterol and cholesterol precursors lathosterol and desmosterol were analyzed by GC-MS (n = 6 per group, means ± SEM). A, Left, The ratio of 24S-OH cholesterol to cholesterol in cortex and hippocampus of control and ABCA1^−B/−B mice. A, Right, Plasma levels of 24S-OH cholesterol. B, Western blot shows cortical CYP46A1 levels (representative of several experiments). C, Confocal images of hippocampal sections from a wild-type mouse show colocalization of the astrocytic marker GFAP (red) and CYP46A1 (green). Scale bar, 10 μm. D, Graphs show ratios of lathosterol to cholesterol and desmosterol to cholesterol in hippocampus and cortex as well as plasma lathosterol and desmosterol levels. Ctx, Cortex; Hipp, hippocampus. *p < 0.05, **p < 0.01 versus control mice.

Figure 7.

Behavioral function in Abca1^−B/−B mice. A, Spontaneous locomotor activity was measured in 3-month-old control and Abca1^−B/−B mice during a 1 h session in an open field chamber. Specific aspects of locomotor activity including horizontal (distance) and vertical (number of rears) activity were analyzed (n = 11–12). Data show means ± SEM B, Acoustic startle response at various startle intensities. C, PPI of acoustic startle measured at prepulse intensities of 72, 74, 78, and 86 dB (n = 11 per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with control mice.

Figure 8.

Morphological and ultrastructural synaptic analysis in Abca1^−B/−B mice. A, Confocal images showing neuronal synaptotagmin immunofluorescence in the cortex of control and Abca1^−B/−B mice. Top images show neuronal synaptotagmin distribution and bottom images show synaptic puncta staining at high magnification. Scale bar, top images: 10 μm; bottom images: 2 μm. B, Transmission electron microscope images of synapses in the cortex of control (left) and Abca1^−B/−B (right) mice. A synapse was identified by a well defined postsynaptic density and at least three docked vesicles (arrowheads). C, The number of synapses was counted in a total area of 2000 μm². Synapses were identified either as excitatory or inhibitory based on morphology (see Materials and Methods). D, The number of perforated synapses, i.e., synapses containing segmented postsynaptic density (arrows). E, The numbers of docked vesicles and the vesicles in the reserve pool were counted in 150 excitatory synapses from each mouse. Data show means ± SEM. *p < 0.05 and **p < 0.01 compared with control mice. n = 5 per group. Scale bar, 500 nm.