Oral supplementation with docosahexaenoic acid and uridine-5'-monophosphate increases dendritic spine density in adult gerbil hippocampus

Abstract

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer's disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA's behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5'-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (>30%) is accompanied by parallel increases in membrane phosphatides and in pre- and post-synaptic proteins within the hippocampus. Hence, oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.

Fig. 1. Oral supplementation with DHA increases dendritic spine density in adult gerbil hippocampus

Eight animals were randomly divided into 4 groups and were supplemented with 0, 50, 100 or 300 mg/kg of DHA daily for 4 weeks. (A) Primary apical dendrites of CA1 pyramidal neurons. (B) Animals supplemented with 100 or 300 mg/kg/day showed increased spine density: a 12 % increase after the 100 mg/kg/day dose (*p = 0.04) and an 18 % increase after the 300 mg/kg/day dose (**p < 0.001 vs. 0 mg/kg/day). n = 16 ~ 20 neurons from 2 animals per group. One-way ANOVA followed by Tukey’s test.

Fig. 2. DHA-induced dendritic spine formation in adult gerbil hippocampus is enhanced by co-supplementation with UMP

Animals received UMP (0.5%), DHA (300 mg/kg) or both daily for 4 weeks; control gerbils received neither. (A) Apical dendrites of CA1 pyramidal neurons. (B) Animals supplemented with DHA exhibited a significant increase in spine density (by 19%, *p = 0.004 vs. Control); those receiving both DHA and UMP exhibited a greater increase (by 36%, **p < 0.001 vs. Control or by 17%, p = 0.008 vs. DHA). n = 20 ~ 25 neurons from 4 animals per group. One-way ANOVA followed by Tukey’s test. (C) The effect of DHA-plus-UMP on spine density was apparent by 1 week after the start of the treatment. The treated groups (total 8 animals) received both UMP (0.5%) and DHA (300 mg/kg) daily for 1, 2, 3 or 4 weeks; the control groups (8 animals) were given only a regular diet. n = 12 ~ 20 neurons from 2 animals per group. Two-way ANOVA followed by Tukey’s test. *p = 0.02; ** p < 0.001.

Fig. 3. Supplementation with DHA or DHA-plus-UMP increases major membrane phospholipids in adult gerbil hippocampus

(A) PC, phosphatidylcholine; (B) PE, phosphatidylethanolamine; (C) PS, phosphatidylserine; (D) PI, phosphatidylinositol. The increases ranged from 26% (PE by DHA) to 160% (PS by DHA-plus-UMP) compared with their controls. n = 4 ~ 7 animals per group including all the animals used for the dendritic spine analysis shown in Fig. 2B. For those animals, the hippocampus was collected from the contralateral hemisphere. The data are shown as percentage of the average of Controls. One-way ANOVA followed by Tukey’s test. * p < 0.05; *** p < 0.001 vs. Controls.

Fig. 4. Supplementation with DHA or DHA-plus-UMP elevates expression of pre-and post-synaptic proteins in adult gerbil hippocampus

(A and B) Expression levels of the post-synaptic proteins PSD-95 (A) and GluR-1 (B) were increased by 42% and 29% with DHA alone, and by 44% and 37% with DHA-plus-UMP, respectively. (C) Expression of the pre-synaptic protein Synapsin-1 was increased by 37% with DHA alone and by 57% with DHA-plus-UMP. (D and E) Expression of cytoskeletal proteins. The level of Actin (D), which can regulate dendritic spine structures, was increased by 60% with DHA alone and by 88% with DHA-plus-UMP, whereas that of -tubulin (E), which is not specifically localized within synaptic structures, was unaffected with any of the treatments. Hippocampi used for this experiment were collected from the contralateral hemisphere in animals used for the dendritic spine analysis shown in Fig. 2B. n = 4 animals per group. The data are shown as percentage of the average of Controls. One-way ANOVA followed by Tukey’s test. * p ≤ 0.05; ** p < 0.01 vs. Controls. See also supplementary information (SI) Fig. 7.

Fig. 5. Oral supplementation with arachidonic acid (ARA) does not increase dendritic spine density in adult gerbil hippocampus

(A) Primary apical dendrites of CA1 pyramidal neurons. (B) Supplementation with ARA did not change spine density in adult gerbil CA1. n = 18 ~21 neurons from 2 animals per group. (C) Relative amounts of phosphatides in the hippocampi obtained from animals treated with 300 mg/kg/day of ARA. The data are shown in percentage of the average of controls for each phosphatide. There was no statistical difference in any phosphatide between the Control and ARA groups. t-test was performed for each phosphatide (the α level was adjusted to .012 for multiple comparisons). PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol. (D) Western blot analysis for pre- and post-synaptic marker proteins in the same hippocampal homogenatesas as those used for the phosphatide assay (β-tubulin was used as a negative control). Supplementation with ARA did not significantly increase the expression of the marker proteins. Syn-1, Synapsin-1. n = 4 animals for each of Control and ARA groups. Tested by multiple t-tests for individual proteins (α = 0.01).

Fig. 6. A model for spine formation by supplementation with DHA and UMP

The diagram shows phosphatidylethanolamine (PE) synthesis as an example. In neurons, DHA is acylated into the sn-2 position of glycerol-3-phosphate (G3P) to form DAG, together with a saturated fatty acid at the sn-1 position. UMP, a precursor of uridine, is metabolized to CTP, the major rate-limiting precursor in membrane phosphatide synthesis. CTP then reacts with phosphoethanolamine to form CDP-ethanolamine. PE is synthesized by combining DAG and CDP-ethanolamine to form the phosphatide in dendritic spines. DHA and uridine may also activate other neuronal mechanisms (e.g., axonal growth and exocytosis) and cell signaling pathways to induce spine formation (see text).