The Involvement of Fatty Acid-Binding Protein 5 in the Blood-Brain Barrier Transport of Docosahexaenoic Acid and Cognition
thesis
posted on 2016-12-15, 02:29 authored by Yijun PanDocosahexaenoic acid (DHA) is an important polyunsaturated
fatty acid (PUFA) required for cognitive function, however the brain has a
limited ability to synthesize its own DHA from the omega-3 (n-3) fatty acid
precursors. Therefore, in order to maintain brain DHA concentrations at a
required level, DHA must be derived from the plasma, and requires transport
across the blood-brain barrier (BBB) to reach the brain parenchyma. To date,
much attention has focused on the mechanisms governing BBB transport of PUFAs
such as DHA, with both passive diffusion and protein-mediated transport being
postulated to be involved in the luminal membrane uptake. Regardless of its
ability to partition across the luminal membrane of brain endothelial cells by
either passive or active processes, the low aqueous solubility of DHA is likely
to limit its cytosolic transfer to the abluminal membrane, necessitating an
intracellular protein to facilitate trafficking of this PUFA across the BBB.
Fatty acid-binding protein 5 (FABP5) is highly expressed at the BBB, and given
that other FABP isoforms have been found to assist fatty acid trafficking in
various tissues and organs, this thesis investigated whether FABP5 facilitates
the BBB transport of DHA, and assists in the maintenance of brain endogenous
DHA levels and cognitive function. In addition, as lower brain DHA levels have
often been observed in post-mortem human brains, this thesis studied whether
BBB transport decreases in Alzheimer’s disease (AD) and whether this is
associated with altered BBB expression of FABP5.
To identify a possible involvement of FABP5 in DHA uptake processes, the binding affinity of FABP5 to DHA was first measured. Human FABP5 (hFAPB5) was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 was assessed using isothermal titration calorimetry. It was demonstrated that DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM), indicating that FABP5 bound avidly to DHA and therefore could be a contributor to cellular uptake processes of DHA.
The putative role of FABP5 in BBB transport of DHA was then assessed in vitro and in vivo. Human immortalized brain microvascular endothelial cells (hCMEC/D3) were transfected with siRNA to downregulate the expression of FABP5, followed by 14C-DHA cellular uptake studies. FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in 14C-DHA cellular uptake. Similarly, 14C-DHA uptake into brain endothelial cells and brain capillaries isolated from FABP5-deficient (FABP5-/-) mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those isolated from their wild-type littermates (FABP5+/+). In addition, FABP5 gene was transfected into COS-7 cells, and the elevated FABP5 expression in COS-7 cells (19.1 ± 6.8%) led to a significant increase in 14C-DHA uptake (29.0 ± 10.4%). Overall, the in vitro findings suggested that the cellular uptake of DHA was dependent on the levels of FABP5 expression.
In addition, an in situ transcardiac perfusion technique (with no bovine serum albumin included in the perfusate) was optimized and validated in C57/BL6 mice and subsequently used to compare the BBB influx rate (Kin) of 14C-DHA between FABP5+/+ and FABP5-/- mice. Using the optimized conditions (a 1 min pre-perfusion followed by 1 min perfusion of 14C-DHA at 10 mL/min), the Kin of 14C-DHA was determined to be 0.04 ± 0.01 mL/g/s. Relative to FABP5+/+ mice, the Kin of 14C-DHA decreased 36.7 ± 12.4% in FABP5-/- mice. The in vivo study confirmed the essential involvement of FABP5 in the BBB transport of DHA.
Given the importance of DHA in cognition and the involvement of FABP5 in the BBB transport of DHA, the impact of FABP5 deletion on endogenous brain DHA levels and cognitive function was then investigated. Cognitive function was assessed in male and female FABP5+/+ and FABP5-/- mice using a battery of memory paradigms, including T-maze spontaneous alternation, Y-maze, novel object recognition, water maze and contextual fear conditioning. FABP5-/- mice exhibited impaired working memory (12.9 ± 3.3% decrease) in T-maze spontaneous alternations. In addition, impaired short-term spatial memory was demonstrated in female FABP5-/- mice, which were not able to distinguish between the novel and familiar arm of the Y-maze. Moreover, FABP5-/- mice required more training to obtain the water maze task than the FABP5+/+ mice, and FABP5-/- mice had a significantly lower discrimination index in the novel object recognition task relative to FABP5+/+ mice, which was close to zero, suggesting impaired episodic memory. In addition, deficits in fear memory formation and spatial memory retrieval ability (in the water maze) were also displayed in FABP5-/- mice. These cognitive deficits were associated with a 27.4 ± 10.3% reduction in endogenous brain DHA levels due to FABP5 genetic deletion. Such results strongly support the hypothesis that FABP5 is essential for maintaining brain DHA levels, and that cognitive deficits observed in FABP5-/- mice are associated with reduced central nervous system (CNS) access of DHA, which is in line with the crucial role of FABP5 in the brain uptake of DHA.
As AD is associated with a reduction in brain levels of DHA, it was hypothesized that there is a reduction in the BBB transport of DHA and that this was associated with lower FABP5 expression at the BBB. Therefore, DHA transport across the BBB was assessed in 8 month-old AD transgenic mice (APP/PS1) and FABP5 expression in isolated cerebral microvessels was measured in these mice, relative to wild-type (WT) mice. Interestingly, a reduction in BBB transport of 14C-DHA (42.1 ± 12.6%) was observed in APP/PS1 mice, and this was associated with a decrease in FABP5 expression (34.5 ± 6.7%) in the isolated microvessels from APP/PS1 mice.
Since impaired DHA transport was observed in AD mice, follow-up investigations were conducted to identify whether long-term (6-month) dietary restriction of DHA increases the vulnerability of APP/PS1 mice to cognitive deficits relative to WT mice. It was hypothesized that the BBB with FABP5-deficiency (observed in AD) would have impaired ability to derive DHA from plasma, and therefore this would result in an exacerbation of the negative effects of DHA dietary restriction. Deficits in short-term spatial memory (Y-maze) were identified only in APP/PS1 mice fed an n-3 fatty acid-depleted diet and not in APP/PS1 mice fed a control diet or WT mice fed a control or n-3 fatty acid-depleted diet. In addition, only the APP/PS1 mice fed the n-3 fatty acid-depleted diet demonstrated impaired episodic memory (novel object recognition). A 34.3 ± 9.0% reduction in endogenous brain DHA levels was also detected in APP/PS1 mice fed an n-3 fatty acid-depleted diet compared to WT mice fed a control diet, suggesting a combined effect of diet and AD transgene. Overall this series of studies demonstrate an association between FABP5-deficiency at the BBB in AD transgenic mice (8 month-old) and increased vulnerability to a 6-month n-3 fatty acid dietary restriction, in line with our previous studies demonstrating that FABP5 facilitates DHA access into the CNS and is important for maintenance of cognitive function.
To identify a possible involvement of FABP5 in DHA uptake processes, the binding affinity of FABP5 to DHA was first measured. Human FABP5 (hFAPB5) was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 was assessed using isothermal titration calorimetry. It was demonstrated that DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM), indicating that FABP5 bound avidly to DHA and therefore could be a contributor to cellular uptake processes of DHA.
The putative role of FABP5 in BBB transport of DHA was then assessed in vitro and in vivo. Human immortalized brain microvascular endothelial cells (hCMEC/D3) were transfected with siRNA to downregulate the expression of FABP5, followed by 14C-DHA cellular uptake studies. FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in 14C-DHA cellular uptake. Similarly, 14C-DHA uptake into brain endothelial cells and brain capillaries isolated from FABP5-deficient (FABP5-/-) mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those isolated from their wild-type littermates (FABP5+/+). In addition, FABP5 gene was transfected into COS-7 cells, and the elevated FABP5 expression in COS-7 cells (19.1 ± 6.8%) led to a significant increase in 14C-DHA uptake (29.0 ± 10.4%). Overall, the in vitro findings suggested that the cellular uptake of DHA was dependent on the levels of FABP5 expression.
In addition, an in situ transcardiac perfusion technique (with no bovine serum albumin included in the perfusate) was optimized and validated in C57/BL6 mice and subsequently used to compare the BBB influx rate (Kin) of 14C-DHA between FABP5+/+ and FABP5-/- mice. Using the optimized conditions (a 1 min pre-perfusion followed by 1 min perfusion of 14C-DHA at 10 mL/min), the Kin of 14C-DHA was determined to be 0.04 ± 0.01 mL/g/s. Relative to FABP5+/+ mice, the Kin of 14C-DHA decreased 36.7 ± 12.4% in FABP5-/- mice. The in vivo study confirmed the essential involvement of FABP5 in the BBB transport of DHA.
Given the importance of DHA in cognition and the involvement of FABP5 in the BBB transport of DHA, the impact of FABP5 deletion on endogenous brain DHA levels and cognitive function was then investigated. Cognitive function was assessed in male and female FABP5+/+ and FABP5-/- mice using a battery of memory paradigms, including T-maze spontaneous alternation, Y-maze, novel object recognition, water maze and contextual fear conditioning. FABP5-/- mice exhibited impaired working memory (12.9 ± 3.3% decrease) in T-maze spontaneous alternations. In addition, impaired short-term spatial memory was demonstrated in female FABP5-/- mice, which were not able to distinguish between the novel and familiar arm of the Y-maze. Moreover, FABP5-/- mice required more training to obtain the water maze task than the FABP5+/+ mice, and FABP5-/- mice had a significantly lower discrimination index in the novel object recognition task relative to FABP5+/+ mice, which was close to zero, suggesting impaired episodic memory. In addition, deficits in fear memory formation and spatial memory retrieval ability (in the water maze) were also displayed in FABP5-/- mice. These cognitive deficits were associated with a 27.4 ± 10.3% reduction in endogenous brain DHA levels due to FABP5 genetic deletion. Such results strongly support the hypothesis that FABP5 is essential for maintaining brain DHA levels, and that cognitive deficits observed in FABP5-/- mice are associated with reduced central nervous system (CNS) access of DHA, which is in line with the crucial role of FABP5 in the brain uptake of DHA.
As AD is associated with a reduction in brain levels of DHA, it was hypothesized that there is a reduction in the BBB transport of DHA and that this was associated with lower FABP5 expression at the BBB. Therefore, DHA transport across the BBB was assessed in 8 month-old AD transgenic mice (APP/PS1) and FABP5 expression in isolated cerebral microvessels was measured in these mice, relative to wild-type (WT) mice. Interestingly, a reduction in BBB transport of 14C-DHA (42.1 ± 12.6%) was observed in APP/PS1 mice, and this was associated with a decrease in FABP5 expression (34.5 ± 6.7%) in the isolated microvessels from APP/PS1 mice.
Since impaired DHA transport was observed in AD mice, follow-up investigations were conducted to identify whether long-term (6-month) dietary restriction of DHA increases the vulnerability of APP/PS1 mice to cognitive deficits relative to WT mice. It was hypothesized that the BBB with FABP5-deficiency (observed in AD) would have impaired ability to derive DHA from plasma, and therefore this would result in an exacerbation of the negative effects of DHA dietary restriction. Deficits in short-term spatial memory (Y-maze) were identified only in APP/PS1 mice fed an n-3 fatty acid-depleted diet and not in APP/PS1 mice fed a control diet or WT mice fed a control or n-3 fatty acid-depleted diet. In addition, only the APP/PS1 mice fed the n-3 fatty acid-depleted diet demonstrated impaired episodic memory (novel object recognition). A 34.3 ± 9.0% reduction in endogenous brain DHA levels was also detected in APP/PS1 mice fed an n-3 fatty acid-depleted diet compared to WT mice fed a control diet, suggesting a combined effect of diet and AD transgene. Overall this series of studies demonstrate an association between FABP5-deficiency at the BBB in AD transgenic mice (8 month-old) and increased vulnerability to a 6-month n-3 fatty acid dietary restriction, in line with our previous studies demonstrating that FABP5 facilitates DHA access into the CNS and is important for maintenance of cognitive function.
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