Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

Abstract

Constitutive macroautophagy involved in the turnover of defective long-lived proteins and organelles is crucial for neuronal homeostasis. We hypothesized that macroautophagic dysregulation in selective brain regions was associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice and employed immunohistochemistry to assess cellular distribution of proteins involved in the selective degradation of ubiquitinated proteins via macroautophagy. Values of the discrimination ratio (DR, a measure of short-term recognition memory performance) in aged mice were significantly lower than those in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in aged mice, there were clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding protein p62, and ubiquitin in neuronal processes predominantly in the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited inverse linear correlations with DR in aged mice (ρ = -0.48 and -0.55, p = 0.044 and 0.018, respectively, Spearman's rank correlation). These findings suggest that increased accumulation of autophagosomes within neuronal processes in selective brain regions is characteristic of aging. The dysregulation of macroautophagy can adversely affect the turnover of aggregate-prone proteins and defective organelles, which may contribute to memory impairment in aged mice.

Fig. 1

Brain sections of aged mice (a–i) are depicted in comparison with those of young mice (j–l). In the hippocampal formation of aged mice, there are scattered clusters of puncta (arrows, higher magnification inset) immunoreactive for microtubule-associated protein 1 light chain 3 (LC3, a), p62 (b), ubiquitin (Ubi, c), microtubule-associated protein 2 (MAP2, e), and synapsin IIa (Syn, f). These immunoreactive puncta (arrows, higher magnification inset) are also present in the olfactory bulb (d) and cerebellar cortex (g). No clusters of immunoreactive puncta are observed on any of the hemi-brain sections immunostained for phosphorylated Tau (p-Tau, h) or synaptophysin (Syp, i). Note that hippocampal formation sections (a–c, e, f, h, i) are of one aged mouse, and olfactory bulb (d) and cerebellar (g) sections are of another aged mouse. By contrast, young mice (j–l, the hippocampal formation of one young mouse) show no (k, l) or sparse (j [arrow], higher magnification inset) clusters of immunoreactive puncta. SO stratum oriens, SP stratum pyramidale, SR stratum radiatum, SL stratum lacunosum-moleculare, SM stratum moleculare, SG stratum granulosum, PZ pleomorphic zone (hilus)

Fig. 2

Double immunofluorescent staining of the hippocampal formation of aged mice shows within clustered puncta co-localization of microtubule-associated protein 2 (MAP2, a, d, h) with microtubule-associated protein 1 light chain 3 (LC3, b, CA1 stratum radiatum), p62 (e, stratum moleculare), and ubiquitin (Ubi, g, CA1 stratum lacunosum-moleculare [upper] and stratum moleculare [lower]). Ubiquitin (j, m, p) is also co-localized within clustered puncta with synapsin IIa (Syn, k [arrows], CA1 stratum lacunosum-moleculare), LC3 (n, CA1 stratum radiatum), and p62 (q, stratum moleculare). c, f, i, l, o, r Each represents a merged image in their respective row, with DAPI-labeled nuclei (blue) in c, f, and i; bar = 20 μm. Note that a–i were obtained from one aged mouse and j–r from another aged mouse

Fig. 3

In the hippocampal formation of all mice studied (22 young and 18 aged), the semiquantitative burden of clustered puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3, a), p62 (b), ubiquitin (c), microtubule-associated protein 2 (MAP2, d), and synapsin IIa (e) each shows a significant linear trend toward greater load in aged mice (p < 0.0001, Chi-square test for linear trend)

Fig. 4

The linear correlation coefficient (ρ) between the numbers of clusters of immunoreactive puncta is shown for each pair of proteins studied in each of three brain regions (i.e., the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex) of 18 aged mice (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Spearman’s rank correlation). Note whether there were clustered puncta immunoreactive for ubiquitin and synapsin IIa in the cerebellar cortex was undetermined due to intense immunoreactivity of these two proteins in the granule cell layer. LC3 microtubule-associated protein 1 light chain 3, Ubi ubiquitin, MAP2 microtubule-associated protein 2

Fig. 5

Scattergraphs with their trend lines show significant inverse correlations between the discrimination ratio (i.e., the ratio of the time spent exploring the novel object over the total amount of time spent exploring both novel and familiar objects during the retention phase of the single-trial object recognition test) as a measure of the short-term recognition memory performance and the number of clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3) and p62 in the hippocampal formation of 18 aged mice (ρ = −0.48 and −0.55, p = 0.044 and 0.018, respectively, Spearman’s rank correlation)