Capsosiphon fulvescens Glycoproteins Enhance Probiotics-Induced Cognitive Improvement in Aged Rats

Abstract

Aging-induced cognitive dysfunction can be regulated by probiotics through bidirectional communication with the brain. This study aimed to investigate whether Capsosiphon fulvescens glycoproteins (Cf-hGP) enhanced probiotic-induced improvement of memory in aged rats and the underlying mechanism in the dorsal hippocampus. Cf-hGP were isolated using lectin resin. Cf-hGP (15 mg/kg/day) and/or Lactobacillus plantarum (L. plantarum) (10⁹ CFU/rat/day) were orally administered once a day for 4 weeks. Co-treatment with Cf-hGP and L. plantarum synergistically improved spatial memory in aged rats, which was overturned by functional blocks of brain-derived neurotrophic factor (BDNF) signaling. Increases in BDNF expression and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation were accompanied by mono- and/or co-administration in the dorsal hippocampus, while c-Jun N-terminal kinase (JNK) phosphorylation and glucose-regulated protein 78 expression were decreased. These synergistic effects were downregulated by blocks of BDNF/Nrf2-mediated signaling. In particular, co-treatment, not mono-treatment, reduced phosphorylation of eukaryotic elongation factor 2 (eEF2) regulated by eEF2 kinase and protein phosphatase 2A. Additionally, co-treatment downregulated the interaction between eEF2 kinase and JNK. These data demonstrated that cognitive impairment in aged rats was synergistically diminished by co-treatment with Cf-hGP and L. plantarum through BDNF-mediated regulation of Nrf2 and eEF2 signaling pathways in the dorsal hippocampus.

Keywords: Capsosiphon fulvescens; aging-induced cognitive dysfunction; psychobiotics.

Figure 1

Effects of Cf-hGP on L. plantarum-induced cognitive improvement in aged rats. Cf-hGP (15 mg/kg) or L. plantarum (10⁹ colony-forming units (CFU)/rat/day) was orally administered once a day for 4 weeks (A). After four weeks, acquisition training was performed (B). The co-treatment with Cf-hGP and L. plantarum synergistically downregulated the latency and upregulated the frequency of platform crossings compared to mono-treatment (C,D). * p < 0.05 versus untreated rats; ^# p < 0.05 versus the rats treated with only Cf-hGP or LP; ^## p < 0.05 versus the rats co-treated with Cf-hGP and LP. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; BDNF, brain-derived neurotrophic factor; WD, withdrawal period.

Figure 2

Expression of mature BDNF and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and c-Jun N-terminal kinase (JNK) in the dorsal hippocampus by co-treatment with Cf-hGP and L. plantarum. Following oral administration of Cf-hGP (15 mg/kg) or L. plantarum (10⁹ CFU/rat/day) once a day for 4 weeks, expression of mature BDNF (mBDNF) and phosphorylation of Nrf2 and JNK in the dorsal hippocampus were measured by immunoblotting (A,B). The co-treatment with Cf-hGP and L. plantarum synergistically increased Nrf2 phosphorylation and mBDNF expression compared to mono-treatment, but JNK phosphorylation was decreased (C–E). * p < 0.05 versus adolescent rats; ^# p < 0.05 versus aged untreated rats; ^## p < 0.05 versus the rats treated with only Cf-hGP or LP. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; p-Nrf2, phosphorylated Nrf2; p-JNK, phosphorylated JNK.

Figure 3

Effects of BDNF signaling activated by co-treatment with Cf-hGP and L. plantarum on Nrf2 and JNK phosphorylation and their interactions. The co-treatment-induced increase in Nrf2 phosphorylation was downregulated by functional blocks with the anti-BDNF antibody (1 µg/µL) (A). According to the double immunofluorescence staining, the co-treatment significantly downregulated the increase in glucose-regulated protein 78 expression in aged rats, which was overturned by blocks with the anti-BDNF antibody (1 µg/µL) (B). Inhibition of Nrf2 with ML385 (an inhibitor of Nrf2, 100 pmol/µL) overturned the co-treatment-induced decrease in JNK phosphorylation and GRP78 expression (C). * p < 0.05 versus adolescent rats; ^# p < 0.05 versus aged untreated rats; ^## p < 0.05 versus the rats co-treated with Cf-hGP and LP. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; recBDNF, recombinant BDNF; dHP, dorsal hippocampus; NeuN, neuronal nuclear antigen; DAPI, 4′,6-diamidino-2-phenylindole; GRP78, glucose-regulated protein 78. The scale bar represents 100 µm.

Figure 4

Regulation of BDNF-mediated phosphorylation of eukaryotic elongation factor 2 (eEF2) by co-treatment with Cf-hGP and L. plantarum. Phosphorylation of eEF2 at threonine 56 was decreased by co-treatment with Cf-hGP and L. plantarum, but not by any mono-treatment (A). Phosphorylation of the eEF2 kinase was upregulated, while eEF2 phosphorylation was downregulated by the co-treatment, which was overturned by functional blocks with the anti-BDNF antibody (1 µg/µL) (B). The co-treatment-induced decrease in eEF2 phosphorylation was downregulated by inhibition of protein phosphatase 2A (PP2A) with LB-100 (an inhibitor of PP2A, 1.5 mg/kg) (C). * p < 0.05 versus untreated rats; ^# p < 0.05 versus the rats co-treated with Cf-hGP and LP. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; eEF2K, eEF2 kinase; p-eEF2K, phosphorylated eEF2K; Thr56, threonine 56; Ser366, serine 366.

Figure 5

Effect of co-treatment with Cf-hGP and L. plantarum on the eEF2 kinase-JNK interaction. JNK was immunoprecipitated using the eEF2 kinase antibody. The interaction between the eEF2 kinase and JNK was downregulated by co-treatment with Cf-hGP and L. plantarum. Further, a significant decrease in JNK phosphorylation was accompanied by an increase in eEF2 kinase phosphorylation. * p < 0.05 versus untreated rats; unpaired t-test. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; eEF2K, eEF2 kinase; dHP, dorsal hippocampus; IP, immunoprecipitation; IgG, immunoglobulin G; Ab, antibody.

Figure 6

Scheme of a proposed mechanism of underlying effects of Cf-hGP on L. plantarum-induced cognitive improvement in aged rats. Chronic administration of Cf-hGP with L. plantarum synergistically attenuated cognitive impairment in aged rats through BDNF-mediated control of Nrf2 and eEF2 signaling in the dorsal hippocampus. Cf-hGP, Capsosiphon fulvescens glycoprotein; LP, Lactobacillus plantarum; dHP, dorsal hippocampus; ER, endoplasmic reticulum.