MiR-324-5p is differentially expressed at select developmental time points
Measuring microRNA expression in whole hippocampal lysate of wildtype mice at various developmental timepoints, we found that miR-324-5p expression varies significantly across development (Fig. 1; Welch’s one-way ANOVA, p < 0.0001; avg n = 11 mice per time point). Following birth, miR-324-5p expression increases until reaching a peak in expression around postnatal day 28 (PD 28). After PD 28, miR-324-5p expression begins to plateau (Dunnett’s T3, ****p(28 vs 1) < 0.0001, *p(28 vs 14) = 0.019, p(28 vs 21) = 0.83, p(28 vs 45–60) = 0.365, p(28 vs 75+) = 0.984). MiR-324-5p is differentially expressed in the hippocampus across development, indicating that this miRNA functions in normal hippocampal development.
Male Mir324 KO mice have reduced body weight in adolescence
Mir324 KO does not lead to significant changes in body weight of adult male or female mice (Parkins et al., 2023). To more thoroughly characterize the effect of Mir324 loss on body weight, we weighed WT and KO mice across development (Fig. 2A,B). Body weight varied significantly by age for both male (2A, two-way ANOVA, ****p(age) < 0.0001) and female (2B, ****p(age) < 0.0001) mice as expected, but genotype only affected male mice (*p(genotype) = 0.0269, **p(interaction) = 0.006). This effect was mainly driven by the period of adolescence, when male Mir324 KO mice were significantly lower in weight than WT mice (***p(PD28) = 0.0008, *p(PD45-60) = 0.0215).
Mir324 KO does not affect hippocampal morphology
We next analyzed gross hippocampal morphology across development. We identified no significant changes in any hippocampal morphology measures by genotype (Fig. 2C-I). The area of the CA1 subregion of the hippocampus varied by age but not genotype (Fig. 2C; two-way ANOVA, ***p(age) = 0.0003, p(genotype) = 0.768, p(interaction) = 0.659), with no significant differences by genotype at any age. The same was found for the area of the dentate gyrus (DG) (Fig. 2D; two-way ANOVA, *p(age) = 0.0188, p(genotype) = 0.953, p(interaction) = 0.548). No effect of genotype was found for any of the other hippocampal measures (H1-5) at any time point (Fig. 2F-I; RM two-way ANOVA, all > 0.05). This suggests that miR-324-5p loss does not affect the gross morphology of the hippocampus.
Mir324 KO leads to a premature reduction in hippocampal dendritic spine density during adolescence
Dendritic spine density varied significantly by age but not genotype (Fig. 3; two-way ANOVA, ***p(age) = 0.0008, p(genotype) = 0.874, p(interaction) = 0.069). Analysis revealed, however, that the pattern of dendritic spine density change across development varied by genotype, with WT mice showing a reduction in dendritic spine density by PD 85+ (Dunnett’s multiple comparisons test, p(14 vs 28) > 0.99, p(14 vs 60) = 0.958, *p(14 vs 85+) = 0.0477) and KO mice showing an earlier reduction in spine density by PD 60 (Dunnett’s multiple comparisons test, p(14 vs 28) = 0.884, **p(14 vs 60) = 0.0073, p(14 vs 85+) = 0.081). The shift in dendritic spine reduction coincided with the observed peak in miR-324-5p expression at PD 28 (Fig. 1), indicating that miR-324-5p plays an important role in normal dendritic spine refinement.
Mir324 KO alters hippocampal PSD95 mRNA and protein expression differentially across development
We next used postsynaptic density protein-95 (PSD95), a synaptic scaffolding protein localized to excitatory synapses (Won et al., 2017), to assess potential changes in excitatory synapse composition across development in Mir324 KO mice. We quantified PSD95 protein (Fig. 4A) and mRNA (Fig. 4B) in hippocampal lysates at select developmental time points. Mir324 KO led to a significant reduction in PSD95 protein expression overall (two-way ANOVA, p(age) = 0.922, **p(genotype) = 0.0038, ***p(interaction) = 0.0002), with the greatest reductions in the Mir324 KO compared with WT at PD 1 (Sidak’s multiple comparisons, **p = 0.0027) and PD 21 (*p = 0.032). PSD95 mRNA was also significantly reduced in KO hippocampi (two-way ANOVA, ***p(age) = 0.0003, ****p(genotype) < 0.0001, **p(interaction) = 0.0034), but this effect was driven by reductions later in development, with significantly lower PSD95 mRNA measured at PD 28 (Sidak’s multiple comparisons, **p = 0.001), PD 45–60 (***p = 0.0006), and PD 85+ (***p = 0.0001). Thus, loss of miR-324-5p dysregulates PSD95 protein and mRNA expression differentially across development.
Kv4.2 protein and mRNA expression in the hippocampus are affected by Mir324 KO in early development
Kv4.2 is an A-type potassium channel targeted by miR-324-5p (Gross et al., 2016). Kv4.2 protein expression varies significantly by age, and genotype affects age-dependent changes (Fig. 5A; two-way ANOVA, ***p(age) < 0.0001, p(genotype) = 0.1222, **p(interaction) = 0.006). Regardless of genotype, Kv4.2 protein expression decreased after PD1. At PD1, but not at any other time point tested, KO hippocampi have significantly reduced Kv4.2 protein expression compared with WT hippocampi (Sidak’s multiple comparisons, **p = 0.0042). Kv4.2 mRNA expression varied by both age and genotype (Fig. 5B; 2-way ANOVA, ****p(age) < 0.0001, ****p(genotype) = 0.0001, ***p(interaction) = 0.0003), with significantly increased expression in Mir324 KO hippocampus at PD1 (Sidak’s multiple comparisons, *p = 0.0347) and PD 14 (****p < 0.0001). On average, Kv4.2 protein expression is increased at PD45-60 in Mir324 KO, corroborating earlier findings that miR-324-5p negatively regulates Kv4.2 expression (Gross et al., 2016; Tiwari et al., 2019). Increased mRNA expression that is not associated with increased protein expression indicates control at the level of Kv4.2 mRNA translation.
Mir324 KO alters hippocampal MAP2 protein and mRNA expression differentially across development
To further elucidate potential mechanisms underlying the differential dendritic spine development in Mir324 KO, we analyzed MAP2 protein and mRNA expression. MAP2 is a microtubule associated protein localized to neuronal dendrites that regulates dendritic spine structure and function (Y. Kim et al., 2020). Interestingly, though we found no effect of age or genotype on MAP2 protein expression in the hippocampus, we did find a significant interaction between age and genotype in protein expression (Fig. 6A; two-way ANOVA, p(age) = 0.144), p(genotype) = 0.64, **p(interaction) = 0.0088). Overall, MAP2 protein expression remained stable across development and did not vary significantly by genotype at any time point (Sidak’s multiple comparisons, all > 0.05). On average, MAP2 protein expression at PD45-60, was increased, in line with earlier findings (Parkins et al., 2023). Though protein expression remained stable across development, MAP2 mRNA expression varied by age (Fig. 6B; two-way ANOVA, ****p(age) < 0.0001, p(genotype) = 0.448, ***p(interaction) = 0.0009), with peak expression at PD1. Notably, this time point coincided with the time point when mRNA expression was significantly increased in Mir324 KO hippocampi (Sidak’s multiple comparisons, **p = 0.001).
Mir324 KO does not alter excitatory synapse composition on CA1 dendritic spines
Dendritic spine density is not a measure of synaptic density, and the reduction in dendritic spine density observed in PD60 Mir324 KO mice may be compensated for by increased synapse formation on dendritic spines. To assess excitatory synapses on dendrites, we performed immunostaining on PD60 hippocampi for the postsynaptic marker PSD95 and presynaptic marker vGlut1 (Fig. 7A,B). Using Manders’ coefficient analysis (Manders et al., 1993), we measured the proportion of colocalized signal between PSD95 and vGlut1 (Manders’ coefficient, Fig. 7C), the proportion of PSD95 that is colocalized with vGlut1 (Manders’ coefficient K1, Fig. 7D), and the proportion of vGlut1 colocalized with PSD95 (Manders’ coefficient K2, Fig. 7E) within the CA1 region. No differences in these measurements were found between Mir324 KO and WT hippocampal neurons (Fig. 7C: unpaired t-test, p = 0.3445; Fig. 7D: unpaired t-test with Welch’s correction, p = 0.784; Fig. 7E: unpaired t-test with Welch’s correction, p = 0.622, n(WT) = 4 mice, n(KO) = 5 mice, an average of 7 images assessed per mouse). To account for any differences in background staining between images, we also measured the Pearson’s correlation coefficient, which is insensitive to background. No differences in Pearson’s correlation were found between genotypes (Fig. 7F: unpaired t-test, p = 0.762, n(WT) = 4 mice, n(KO) = 5 mice, an average of 7 images were analyzed per mouse). Overall, these results suggest that loss of miR-324-5p does not affect excitatory synapse composition on dendritic spines. Example images of immunostained hippocampi are shown in Fig. 7A-B.