Altered synaptic marker abundance in the hippocampal stratum oriens of Ts65Dn mice is associated with exuberant expression of versican

Abstract

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.

Figure 1. Synaptic protein measurement in LMC and Ts65Dn hippocampal extracts

The synaptic proteins SNAP-25 (A), synaptophysin (B), PSD-95 (C) and the astrocyte protein GFAP (D) were measured by ELISA in 5- and 20-month-old LMC and Ts65Dn hippocampal extracts. At 5 months of age, n = 7 or 8 mice per genotype; at 20 months of age, n = 6 mice for each genotype. For each ELISA, values for LMC and Ts65Dn hippocampus at the same age were compared with a Student's t test; *P<0.05, **P<0.01.

Figure 2. Synaptic protein measurement in LMC and Ts65Dn hippocampal region extracts

Hippocampal regions were microdissected from 200 μm hemibrain sections from 20 months old LMC and Ts65Dn mice between Bregma −1.0 mm to Bregma −2.3 mm (A; black lines indicate the boundaries of the sections) and SO (stratum oriens, black outline), DG (dentate gyrus, red outline) and remainder of the hippocampus (Rest of HC, blue outline) were microdissected (B). The synaptic proteins SNAP-25 (C), synaptophysin (D), PSD-95 (E) and astrocyte protein GFAP (F) were measured by ELISA (n = 5 mice for each genotype). For each ELISA, values for LMC and Ts65Dn subregions were compared with a Student's t test; *P<0.05. Images from (A) and (B) adapted from (Paxinos and Franklin, 2001).

Figure 3. RAWM performance in aged LMC and Ts65Dn mice

20-month-old LMC and Ts65Dn mice (n = 7 or 8 mice per genotype) received 12 RAWM trials (four blocks of three trials) for each day tested. Data were analysed with a two-way repeated measures ANOVA and Bonferroni pairwise comparison; *P<0.05 for Ts65Dn compared with LMC at the same time point. For each genotype, errors for blocks 2–12 were compared with errors from block 1 with a one-way ANOVA with Bonferroni pairwise comparison test;ˆP<0.01.

Figure 4. Versican V2 abundance and processing in LMC and Ts65Dn hippocampal extracts

Immunoblots of hippocampal extracts from mice at 5 months of age, n = 7 mice per genotype; and mice at 20 months of age, n = 6 mice per genotype. NIVNSE refers to the ADAMTS-derived N-terminal fragment of versican V2. Mean densitometric values were normalized to GAPDH (data not shown) and then expressed as a percentage of LMC. For each age, values for LMC and Ts65Dn hippocampus were compared with a Student's t test; *P<0.05.

Figure 5. Versican V2 abundance in LMC and Ts65Dn hippocampal region extracts

Immunoblots from 20-month-old LMC and Ts65Dn hippocampal regions (n = 5 mice for each genotype). The mean versican V2 densitometric value for each sample was normalized to GAPDH (data not shown) and then expressed as a percentage of LMC. For each hippocampal region, the LMC and Ts65Dn values were compared with a Student's t-test; *P<0.05.

Figure 6. Correlation analysis between versican V2 in hippocampal region extracts and RAWM performance for LMC and Ts65Dn mice

Correlation between versican V2 immunoreactivity and the number of errors committed in the final block (block 12) of the RAWM for stratum oriens (A), dentate gyrus (B), and rest of hippocampus (C). Linear regression analysis was performed to determine the significance of the r² value (see the text).

Figure 7. PNNs in LMC and Ts65Dn stratum oriens

Representative images of WFA-positive PNNs in LMC (A) and Ts65Dn (B) SO (stratum oriens) and SP (stratum pyramidale). Individual PNNs were measured and the mean intensity and area determined (n = 141 LMC PNNs and n = 125 Ts65Dn PNNs). PNNs were then categorized based on their mean intensity per area (C). Note that category 1 indicates the lowest mean intensity/area. Mean intensity/area values for measured PNNs were also compared (D). PNNs were also categorized by area (E; category 1 is smallest area) and the values for area of PNNs was compared (F). (C) and (E) were analysed with Cochran–Armitage test for trend (see the text for details). *P<0.05 and **P<0.01 by Mann–Whitney U test. (A, B) Scale bar: 100 μm.