Oral treatment of 4-methylumbelliferone reduced perineuronal nets and improved recognition memory in mice

Abstract

Hyaluronan (HA) is a core constituent of perineuronal nets (PNNs) that surround subpopulations of neurones. The PNNs control synaptic stabilization in both the developing and adult central nervous system, and disruption of PNNs has shown to reactivate neuroplasticity. We investigated the possibility of memory prolongation by attenuating PNN formation using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis. Adult C57BL/6 mice were fed with chow containing 5% (w/w) 4-MU for 6 months, at a dose ~6.7 mg/g/day. The oral administration of 4-MU reduced the glycosaminoglycan level in the brain to 72% and the spinal cord to 50% when compared to the controls. Spontaneous object recognition test (SOR) performed at 2, 3, 6 and 7 months showed a significant increase in SOR score in the 6-months treatment group 24 h after object presentation. The effect however did not persist in the washout group (1-month post treatment). Immunohistochemistry confirmed a reduction of PNNs, with shorter and less arborization of aggrecan staining around dendrites in hippocampus after 6 months of 4-MU treatment. Histopathological examination revealed mild atrophy in articular cartilage but it did not affect the motor performance as demonstrated in rotarod test. In conclusion, systemic oral administration of 4-MU for 6 months reduced PNN formation around neurons and enhanced memory retention in mice. However, the memory enhancement was not sustained despite the reduction of PNNs, possibly due to the lack of memory enhancement training during the washout period. Our results suggest that 4-MU treatment might offer a strategy for PNN modulation in memory enhancement.

Keywords: Extracellular matrix; Hyaluronan; Memory; Neuroplasticity; Perineuronal net.

Fig. 1

The sample (a) and choice (b) phases of the SOR test used for the memory retention analysis.

Fig. 2

The effect of flavor on the amount of consumed chow and weight of animals. Mice were fed with the control or 5% (w/w) 4-MU chow with a chocolate, orange or banana flavor ad libitum for the 28 days. (A) Remarkable enhancement of consumed chow was observed for banana flavor compared with all other groups. (One Way ANOVA; Dunn's Method; p*** = 0.0001). (B) The corresponding amount of consumed 4-MU. (C) The weight of the animals did not differ in all groups. n = 4 per group. (One Way ANOVA; Dunn's Method; p*** = 0.0003).

Fig. 3

4-MU treatment has led to a reduction of GAGs in the CNS. Total GAG content (ug/mg of wet weight) was normalized to the quantity in the control treated mice. 4-MU treatment has led to a significant reduction of total GAG both in the spinal cords (from 100% to 49.93% ± 3.99%, p = 0.0127) and the brains (from 100% to 72.46% ± 4.51%, p = 0.0300). There is a stronger reduction in the spinal cord than in the brain (50.07% Vs 27.54%). n = 3, Student t-test, two tailed paired.

Fig. 4

The spontaneous alternation (SA) and activity test after 2 and 6 months of the 4-MU treatment. (A) SA as well as an activity test did not show any significantly changes after the administration of the 4-MU treatment compared to the control animals. (B) SOR test was done after 2/3/6 month of the 4-MU treatment and then after 1 month (washout). A significant enhancement of SOR score was found in the 3 h delay in animals treated with 4-MU after 2 months compared to the wash-out group and in 24 h delay after 6 months of treatment compared to control and wash out group of animals. SOR score decreased to the control level after 1 month wash-out, which suggests that the effect of 4-MU is temporal. *p < 0.05; **p < 0.01, One Way ANOVA; Tukey’s multiple comparison test.

Fig. 5

(A) Representative images of immunofluorescence staining of WFA in CA1-CA3 of the hippocampal area in the control, 4-MU treated for 6 months and 4-MU treated animals after 1 month of the wash-out. (B) Intensity of WFA staining and total number of WFA positive cells determined in the hippocampal area (CA1–CA3). A significant decline of the WFA intensity staining was found after 6 months of 4-MU treatment and it persisted after 1 month of the wash-out when compared with the control. n = 4 per group, ***p < 0.001; one-way ANOVA test. WOE – wash-out effect. Scale: 50 µm.

Fig. 6

(A–C) Illustrations of aggrecan stained PNNs in neurons in the hippocampal CA2/CA3 area in the (A) control, (B) 4-MU after 6 months of treatment and (C) WOE group. (D–F) Examples of PNN tracing in the (D) control, (E) 4-MU and (F) WOE. Arrows in A, B, C depict PNNs of neurons traced in D, E, F. WOE-washout effect. Scale: (A–D) 50 µm, (E, F): 20 µm.

Fig. 7

Morphological parameters of PNNs in the hippocampal CA2/CA3 area in the control, 4-MU after 6 months of treatment, and WOE group. n = 4 per group, 40 neurons per animal, ***p < 0.001, ** p < 0.01, *p < 0.05, Kruskal Wallis test, followed by Dunn's multiple comparisons test.

Fig. 8

mRNA expression of selected genes after(A) 6 months of 4-MU treatment and (B) 1 month of 4-MU wash-out. Significant changes of expression were observed in neurocan (ncan), glutamate ionotropic receptor AMPA type subunit 2 (gria2), synaptophysin (syp), (CD44) with 6 months of treatment, and in hyaluornan synthase 3 (has3), ncan and CD44 in the WOE group when compared with the control group. n(control) = 4, n(4-MU) = 5, n(WOE) = 4. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ****p ≤ 0.0001 vs. control, one-way ANOVA; Student-Newman-Keuls Method.

Fig. 9

Alcian blue staining of the articular cartilage. (A, C) Articulation in normal bones is covered by standard thick hyaline cartilages. (B, D) Atrophy of the hyaline cartilage with thin bones, and trabecula with residual, intermittent regenerating hyaline cartilages are observed after 4-MU treatment. Scale bar: A,B: 100 µm, C,D: 10 µm.

Fig. 10

(A) Grip test and (B) rotarod test. (C) The results between control and treated group didn’t show any changes in the strength of the forelimbs period neither motor functions after 6 months of treatment. n = 4 in control and n = 8 in 4-MU group.