Single-neuron analysis of aging-associated changes in learning reveals impairments in transcriptional plasticity

Abstract

The molecular mechanisms underlying age-related declines in learning and long-term memory are still not fully understood. To address this gap, our study focused on investigating the transcriptional landscape of a singularly identified motor neuron L7 in Aplysia, which is pivotal in a specific type of nonassociative learning known as sensitization of the siphon-withdraw reflex. Employing total RNAseq analysis on a single isolated L7 motor neuron after short-term or long-term sensitization (LTS) training of Aplysia at 8, 10, and 12 months (representing mature, late mature, and senescent stages), we uncovered aberrant changes in transcriptional plasticity during the aging process. Our findings specifically highlight changes in the expression of messenger RNAs (mRNAs) that encode transcription factors, translation regulators, RNA methylation participants, and contributors to cytoskeletal rearrangements during learning and long noncoding RNAs (lncRNAs). Furthermore, our comparative gene expression analysis identified distinct transcriptional alterations in two other neurons, namely the motor neuron L11 and the giant cholinergic neuron R2, whose roles in LTS are not yet fully elucidated. Taken together, our analyses underscore cell type-specific impairments in the expression of key components related to learning and memory within the transcriptome as organisms age, shedding light on the complex molecular mechanisms driving cognitive decline during aging.

Keywords: gene expression; molecular biology of aging; neuroscience; senescence.

FIGURE 1

Overview of single‐neuron analysis of aging‐associated changes in learning. (a) Schematic of the known components of the gill siphon withdrawal reflex in Aplysia abdominal ganglia adopted from Kupfermann et al. (1974). (b) Age groups 8 months (Age 1), 10 months (Age 2), 12 months (Age 3). (c) Bar graphs showing the average duration of siphon withdrawal from the stimulus to the time the siphon begins to relax before (Pre) and 24 h after (Test) long‐term sensitization training (LTS) or no shock control in three age groups. The number of animals used for analysis is shown in the bar graphs. Data was first log transformed and then a three‐way ANOVA was performed followed by individual post hoc comparisons. There was a significant Pre versus Post × shock versus control × age group three‐way interaction overall and multiple significant individual comparisons indicated in the figure (**p < 0.001, ****p < 0.0001, NS: nonsignificant. Error bars are SEM, see Table S1). (d) Schematic representation of the workflow for single L7M isolation to RNAseq from trained (short‐term and long‐term sensitization) and untrained animals age groups 1–3. See also related Figure S1 and Table S1.

FIGURE 2

RNAseq analysis of L7MN reveals specific changes in the expression of mRNAs and long‐noncoding RNAs (lncRNAs) following STS and LTS training. Venn diagrams showing (the numbers indicate unique and common differentially expressed genes (DEGs) in 8‐month‐old animals. (a) Upregulated, (b) downregulated in response to short‐term sensitization, STS and long‐term sensitization, LTS (p <0.05; the numbers indicate unique and common DEGs) (see Figures S2–S6, Table S2). Venn diagrams showing differentially expressed lncRNAs (c) upregulated, (d) downregulated in response to STS and LTS (p <0.05). Differentially expressed genes are ranked in a volcano plot according to their statistical‐log2 p (y‐axis) and their relative abundance ratio (log2 fold change) between up‐ and downregulated (x‐axis). Red dots indicate significantly regulated genes (false discovery rate, <0.01; s0 = 1; p <0.05) (see Table S2). Volcano plots of (e). Control versus STS DEGs, (f) Control versus LTS DEGs, (g). LTS versus STS DEGs (see Table S2). Heatmaps showing the normalized and scaled expression values of the top 50 differentially expressed genes when ranked by p‐value. The color gradient from green to red represents high to low expression levels across the samples. The genes are ordered by hierarchical clustering using Euclidean distance and complete clustering method while the samples are ordered by condition, (h) STS versus Control, (i) LTS versus Control, (j) LTS versus STS (see Table S2). qPCR validation of selected candidates from RNAseq data, (k) lncRNAs, (l) mRNAs. Relative gene expression levels are exhibited as the mean fold change, with error bars showing the SEM. One‐way ANOVA followed by Tukey's post hoc test. N = 4, p values are shown in the bar graphs (see Table S2).

FIGURE 3

RNAseq analysis of L7MN from 10‐ and 12‐month‐old Aplysia following STS and LTS training. Venn diagram showing Age 2 DEGs (a) upregulated (b) downregulated in response to STS and LTS (p <0.05). Differentially expressed genes are ranked in a volcano plot according to their statistical‐log2 p‐value (y‐axis) and their relative abundance ratio (log2 fold change) between up‐ and downregulated (x‐axis). Red dots indicate significantly regulated genes (false discovery rate, <0.01; s0 = 1; p <0.05) (see Table S3). Volcano plots of (c) age 2 Control versus STS DEGs, (d) age 2 Control versus LTS DEGs. Venn diagram showing DEG lncRNAs (e) upregulated (f) downregulated in response to STS and LTS (p <0.05) (see Table S3). Venn diagram showing age 3 DEGs. (g) Upregulated (h downregulated in response to STS and LTS (age 3; p <0.05) (see Table S3). Volcano plots of (I) Age 2 Control versus STS DEGs. (j) Age 2 Control versus LTS DEGs (see Table S3). Venn diagram showing DEG lncRNAs (k) upregulated (l) downregulated in response to STS and LTS (age 3; p <0.05) (see Table S3). qPCR validation of the RNAseq data (m) Age 2 lncRNAs, (n) Age 2 mRNAs, (o) Age 3 lncRNAs, (p) Age 3 mRNAs. Relative gene expression levels are shown as the mean fold change, with error bars showing the SEM. One‐way ANOVA followed by Tukey's post hoc test. N = 4, p values are shown in the bar graphs (see Table S3).

FIGURE 4

Analysis of aging‐associated changes in L7MN. RNAseq data from untrained animals (used to generate Figures 2 and 3) were independently compared across the three age groups. Venn diagrams showing comparison of upregulated DEGS (a), downregulated DEGs (b), upregulated lncRNAs (c), and downregulated lncRNAs (d) (p < 0.05) (see Table S4). (e–g) Differentially expressed genes compared to different age groups are ranked in the volcano plots according to their statistical‐log2 p‐value (y‐axis) and their relative abundance ratio (log2 fold change) between up‐ and downregulated (x‐axis). Red dots indicate significantly regulated genes (false discovery rate, <0.01; s0 = 1; p <0.05) (see Table S4). Reanalysis of qPCR candidates from different age groups (see Figures 2 and 3), (h, i) at basal condition, (j, k) in response to short‐term sensitization, (l, m) in response to long‐term sensitization. Relative gene expression levels are shown as the mean fold change, with error bars showing the SEM. One‐way ANOVA followed by Tukey's post hoc test. N = 4, p values are shown in the bar graphs (see Table S4).

FIGURE 5

Gene expression analysis of R2 and L11 MN neurons following STS and LTS training. (a) Schematic representation of the workflow for single R2 and L11 neuron isolation and qPCR analysis from trained (STS and LTS), and untrained control Aplysia from the three age groups. (b–g) Analysis of qPCR candidates in R2 across different age groups (see Table S5). (h–m) Analysis of qPCR candidates in L11 across different age groups. Relative gene expression levels are shown as the mean fold change, with error bars showing the SEM. One‐way ANOVA followed by Tukey's post hoc test. N = 5, p values are shown in the bar graphs (see Table S5).

FIGURE 6

Validation of RNA seq data by qPCR for lncRNAs and its cis pair (± 200 kb). Two lncRNAs and their protein coding mRNAs cis pairs from age1 RNA Seq are examined. Validation of the RNA seq data were done by qPCR for STS and LTS for all three age groups. (a–f) Schematic representation of the location of lncRNAs and its mRNA pair in the genomic region for Cis Pair1 and 2. (b, g) RNASeq data of Cis pair1 and 2 for age1 The Cis pair 1 is non coding RNA lnc78793 (LOC118478793) and uncharacterized protein coding gene mRNA5924 (LOC 101855924) as its Cis pair1. The Cis pair‐2 is non coding RNA is lnc78412 (LOC118478412) and mRNA of tyrosine‐protein kinase hopscotch (LOC101861981) as its cis pair. (c–j) qPCR data of the relative expression of lncRNAs and its cis pair for STS and LTS compared to control for Age 1 (c, h), Age 2 (d, i) and Age 3 (e, j) (see Table S6).