Differences in Gene Expression in the Hippocampus of Aged Rats Are Associated with Better Long-Term Memory Performance in a Passive Avoidance Test

Abstract

Microarray analysis was used to identify genes differentially expressed in the hippocampus of aged rats showing diverse long-term (3 and 6 h) spatial–associative memory performance in a single-trial inhibitory avoidance task. The transcription of 43 genes (including genes functionally linked to signal transduction, cell growth and differentiation, translation, energy metabolism, and nucleic acid processing) was significantly upregulated in good- versus bad-performing animals, whereas that of 18 genes (including genes functionally linked to transcription, cell growth and differentiation, apoptosis, and protein transport) was significantly downregulated in good- versus bad-performing animals. The differential expression of 14 of these genes was confirmed by real-time polymerase chain reaction.

Introduction

B rain aging is associated with both short-term and long-term memory impairments that may lead to significant cognitive decline.¹ The formation of long-term declarative memory is especially affected and shows large variability among individuals.² The genetic bases for such variability are not yet fully understood. Recent studies have shown that the expression of selected genes, included in the functional categories of protein modification, transcription/translation, signal transduction, and neurotransmission, is differentially regulated in the hippocampus of aged rats showing a better performance in spatial memory (a form of declarative memory) tested with the Morris water maze paradigm.³

In the present work, we have studied the hippocampal genetic profile associated with better performance in spatial–associative memory using an aversive learning paradigm, the inhibitory avoidance (IA) task, in aged rats. The animals were separated in good responders (GR) and bad responders (BR) according to their performance in the task, and hippocampal gene expression was analyzed with microarray analysis in both groups. To validate the expression pattern obtained, RNA samples from the two experimental groups were then examined by means of real-time polymerase chain reaction (PCR).

Materials and Methods

Locomotor and inhibitory avoidance tasks

Female Wistar aged (26–27 months old, n = 12) rats were used for this study. The animals were treated in accordance with the European Community guidelines on animal care, and the experimental protocols were approved by the Ethical Committee of the University of Bologna. Rats were housed 4–5 subjects per cage with ad libitum access to food and water, and maintained on a 12-h light/dark cycle from 8:00 am to 8:00 pm at constant temperature (24 ± 1°C) and humidity (50 ± 5%). The IA test was performed by using an IA chamber (Med Associated Inc., USA) as previously described.⁴ To exclude rats with motor impairments, the animals were previously individually tested for locomotor activity and the following parameters were calculated: (1) the time spent in the corner spaces (t corner); (2) the number of grids crossed by the front part of the rat body (f crossings); and (3) the number of rearings on the forelimbs (f rearings). The animals were tested 3 and 6 h after the administration of a foot shock (0.6 mA for 2 s). According to the performance displayed in the tests, they were separated into two groups: (1) GR, which obtained retention times above 100 sec in both tests, and (2) BR, which obtained retention times below 100 s in both tests. Immediately after the last test, the rats were anesthetized deeply and their hippocampi were removed and frozen in dry ice.

Microarray analysis

Total hippocampal RNA from each animal (n = 6 for each group) was extracted by means of the RNeasy Lipid Tissue kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. Linear amplification of RNA was carried out using an Amino Allyl MessageAmp aRNA Kit (Ambion, Austin, TX). Briefly, double-stranded complementary DNA (cDNA) was synthesized from 1 μg of total RNA, then in vitro transcription was performed in the presence of aminoallyl uridine-5′-triphosphate (UTP) to produce multiple copies of aminoallyl-labeled complementary RNA (aRNA). aRNA was then labeled with N-hydroxy succinimide esters of Cy5 for aRNA from the GR group and Cy3 for aRNA from the BR group. Both labeled aRNAs were co-hybridized with an OciChip Rat 10K microarray (Ocimum Biosolutions, Hyderabad, India) containing 9715 spotted rat sequences. Following hybridization, microarrays were washed and analyzed with GenePix Pro 6.0 software connected to a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). The ratio of medians of the pixel fluorescence intensity between Cy5 (GR sample) and Cy3 (BR sample) was considered for each feature; then data extracted from the images were transferred to the software package Acuity 4.0 (Molecular Devices) for normalization and quality filtering. We then applied two additional constraints to the data: (1) the ratio of the medians had to be >1.4 (upregulated gene) or <0.71 (downregulated gene) in at least four of the six DNA microarrays and (2) these genes had to show a p value < 0.05 as calculated by a one-sample t-test. Selected genes were classified according to Gene Ontology category “Biological Process” using the Rat Genome Database (http://rgd.mcw.edu).

Real-time PCR

To validate the expression pattern obtained by array hybridization, RNA samples from the two experimental groups were examined by means of real-time PCR. To this purpose, a subset of differentially expressed genes (13 upregulated and 3 downregulated) was selected. The PCR primer sets were designed using Beacon Designer Software and β-glucuronidase (Gusb) was used as a reference for normalization. Quantitative PCR was performed by means of an iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA), with 200–400 nM for each primer and 10–20 ng of cDNA (RNA equivalent) for each gene. Cycle thresholds were averaged for each gene, and relative quantification was performed according to the Pfaffl method.⁵ Specificity of PCR products was verified by means of melt–curve analysis.

Results

No significant differences were found in GR versus BR in the time spent in the four corners of the open-field arena (t corner, 342 ± 14 s vs. 364 ± 33 s, p > 0.05), in the number of grids crossed by the front part of the rat body (f crossing, 192 ± 8 vs. 201 ± 22, p > 0.05), and in the number of wall-supported rearings on the forelimbs (f rearings, 36 ± 4 vs. 30 ± 3, p > 0.05). Mean retention times obtained in the IA test were 351 ± 102 s (GR) vs. 38 ± 16 s (BR) 3 h after the training, and 375 ± 88 s (GR) vs. 51 ± 18 s (BR) 6 h after the training.

Microarray analysis showed that the transcription of 43 genes was upregulated and that of 18 genes was downregulated in the hippocampus of GR versus BR rats. According to Gene Ontology category “Biological Process” using the Rat Genome Database (http://rgd.mcw.edu), differentially expressed genes were assigned to the 12 functional categories shown in Table 1. A subgroup of 16 genes representative of the majority of functional categories was selected for validation of the microarray analysis by means of real-time PCR as indicated in Table 1. This analysis confirmed the differential expression of all selected genes, with the exception of Ddx and Serpinb5.

Table 1.

Genes Differentially Expressed in the Hippocampus of GR versus BR Rats

Biological process	Gene name	Gene symbol	Gene ID	Fold change (mean ± SD)	p value (t-test)
Cell growth and differentiation
	Transient receptor protein 6^a	Trpc6	NM_053559	1.91 ± 0.79	0.0254
	Nel-like 2 (chicken)^a	Nell2	NM_031070	1.57 ± 0.14	0.0001
	Plectin 1	Plec1	NM_022401	1.37 ± 0.29	0.0326
	Shroom family member 2	Shroom2	NM_001047893	1.34 ± 0.27	0.0379
	Cytidine 5′-triphosphate synthase	Ctps	XM_233467	0.27 ± 0.11	0.0015
	Breast cancer metastasis-suppressor 1	Brms1	NM_001009605	0.59 ± 0.20	0.0142
Signal transduction
	Rho family GTPase^a	Rnd2	NM_0010110953	1.51 ± 0.15	0.0002
	Dynamin binding protein^a	Dnmbp	XM_219860	1.87 ± 0.33	0.0018
	Insulin receptor 3	Irs3	NM_032074	1.38 ± 0.28	0.0227
	F-box and WD40 domain protein 2	Fbxw2	NM_001107835	1.39 ± 0.31	0.0301
	Ras-GTPase-activating prot. SH3-dom. bind.protein	G3bp1	XM_340802	0.67 ± 0.18	0.0108
	Traf type zinc finger domain containing 1	Trafd1	XM_341086	0.70 ± 0.12	0.0071
Transcription
	Neurogenic differentiation 6^a	Neurod6	XM_575489	1.51 ± 0.43	0.0332
	Suppressor of variegation 4–20 homolog 2	Suv420h2	XM_218221	1.39 ± 0.20	0.0050
	Cyclin-dependent kinase 7	Cdk7	X83579	1.32 ± 0.28	0.0394
	Sirtuin 5	Sirt5	NM_001004256	0.46 ± 0.11	0.0087
	Maf1 homolog	Maf1	NM_0010114085	0.59 ± 0.20	0.0142
Translation
	Ribosomal protein S6^a	Rps6	NM_017160	1.52 ± 0.27	0.0023
	60S Ribosomal protein L12	Rpl12	X53504	1.49 ± 0.29	0.0066
	Ribosomal protein L10	Rpl10	NM_031100	1.49 ± 0.17	0.0066
	Ribosomal protein L32	Rpl32	NM_013226	1.41 ± 0.34	0.0289
	Finkel-Biskis-Reilly mur. Sarc. virus ubiq. exp.	Fau	NM_001012739	1.40 ± 0.16	0.0011
	Mitochondrial ribosomal protein L41^a	Mt-rpl41	XM_576439	1.39 ± 0.22	0.0056
	Ribosomal protein S24	Rps24	NM_031112	1.36 ± 0.29	0.0321
	Ribosomal protein large P1	Rplp1	NM_001007604	1.36 ± 0.21	0.0066
	Ribosomal protein L22	Rpl22	NM_031104	1.40 ± 0.29	0.0182
	Ubiquitin A-52 ribosomal protein	Uba52	NM_031687	1.33 ± 0.26	0.0305
Protein transport
	Atg4 Autophagy-related homolog B ^a	Atg4	NM_001025711	0.47 ± 0.13	0.0117
	DnaJ (Hsp40) homolog, subfamily C, member 14	Dnajc14	NM_53690	0.61 ± 0.15	0.0850
	Lysine deficient protein kinase 4	Wnk4	NM_175579	0.61 ± 0.12	0.0019
	Phosphatidylinositol transfer protein membrane-1	Pitpnm1	NM_0010083	0.53 ± 0.09	0.0002
	Solute carrier family 4, member 1, adaptor protein	Slc4a1ap	XM_233883	0.42 ± 0.09	0.0008
	Trafficking protein, kinesin binding 2 ^a	Trak2	NM_133560	0.56 ± 0.17	0.0204
	Transmembrane emp24 protein transport dom. 9	Tmed9	NM_001009703	0.63 ± 0.24	0.0224
Energy metabolism
	Cytochrome c oxidase subunit VIIb (cox7b)^a	Cox7b	NM_182819	1.49 ± 0.28	0.0218
	Malate dehydrogenase 1B, NAD soluble	Mdh1b	XM_237203	1.41 ± 0.25	0.0060
	Succinate dehydrogenase complex subunit B^a	Sdhb	XM_216558	1.38 ± 0.30	0.0272
Apoptosis
	Protein disulfide isomerase associated-3	Pdia3	NM_017319	1.40 ± 0.17	0.0012
	Apaf1 interacting protein^a	Apip	XM_215785	1.72 ± 0.55	0.0180
	Nonmetastatic cell espressed protein 3	Nme3	NM_053507	1.46 ± 0.36	0.0457
	Cytochrome c somatic	Cyc	NM_012839	0.62 ± 0.17	0.0138
	Fermitin family homolog 3	Fermt3	XM_219540	0.46 ± 0.15	0.0095
Nucleic acid processing
	Lsm7 homolog, U6 small nuclear RNA associated	Lsm7	NM_001108732	1.39 ± 0.31	0.0459
	DEAD (asp-Glu-ala-Asp) box polypeptide 1^b	Ddx	NM_053414	1.64 ± 0.50	0.0224
	Lsm8 homolog, U6 small nuclear RNA associated	Lsm8	XM_216102	1.43 ± 0.29	0.0128
Protein processing
	Presenilin stabilization factor-like^a	Aph1b	NM_001047090	1.90 ± 0.53	0.0077
	Aspartyl aminopeptidase ^a	Dnpep	NM_001024879	0.39 ± 0.16	0.0278
Lipid binding
	NSFL1 (p97) cofactor (p47)	Nsfl1c	NM_031981	1.37 ± 0.21	0.0075
Immune response
	Serine proteinase inhibitor, clade B, member 5^b	Serpinb5	NM_057108	1.61 ± 0.36	0.0068
Others
	Microtubule-actin crosslinking factor 1	Macf1	NM_001135758	1.43 ± 0.32	0.0203
	G protein-coupled receptor 108	Gpr108	NM_199399	1.37 ± 0.22	0.0076
	Similar to MGC108823 protein	RGD1559715	XM_574157	1.54 ± 0.26	0.0027
	Similar to NGF-binding Ig light chain	RGD1564462	XM_578320	2.13 ± 1.04	0.0317
	RGD1560662	RGD1560662	XM_579773	1.50 ± 0.32	0.0102
	Similar to SG2 gene	RGD1562200	XM_343790	0.46 ± 0.15	0.0054

Genes whose differential expression was validated by real-time PCR. Downregulated genes are reported in italics.

Genes whose differential expression was not validated by real-time PCR.

Abbreviations: GR, Good responders; BR, bad responders; GTPase, guanosine triphosphatase; NAD, nicotinamide adenine dinucleotide; NGF, nerve growth factor; Ig, immunoglobulin.

Discussion

Present data indicate that the transcription of 61 genes is differentially regulated in aged rats showing a better long-term associative-spatial memory performance. Interestingly, three upregulated (Trpc6, Nell2, Plec1) genes and one downregulated (Ctps) gene included in the cell growth and differentiation category are involved in different aspects of synaptic plasticity. Trpc6 is a G-protein-coupled receptor-operated Ca²⁺ channel, which promotes dendritic growth via the Ca²⁺/calmodulin-dependent kinase IV–cAMP-response-element binding protein (CaMKIV-CREB) pathway⁶; Plec1 is a cytoskeletal cross-linking protein, which interacts with all the three major groups of cytoskeletal proteins (actin filaments, microtubules, and intermediate filaments)⁷; Nell2 is a target of protein kinase C (PKC) that mediates various signaling pathways⁸; Ctps binds tubulin.⁹ Also the signal transduction category includes differentially expressed genes related to cytoskeleton rearrangements, such as Dnmbp,¹⁰ the Rho family guanosine triphosphatase (GTPase) Rnd2,¹¹ and Irs3.¹² These data are consistent with the concept that 2–6 h after a memory training, de novo gene transcription occurs, mainly concerning structural genes required for the synaptic remodeling underlying long-term memory formation.¹³ Accordingly, we have recently found that synaptic remodeling in the hippocampal CA1 region of aged rats correlates with better memory performance in IA.⁴

We found many differentially expressed genes related to transcription and translation. Among them, the upregulated gene Neurod6 is a member of the basic helix–loop–helix transcription factor family involved in neuronal differentiation and maturation.¹⁴ Many ribosomal proteins resulted upregulated in GR. The specific role of each ribosomal protein during ribosome biogenesis and protein synthesis is still uncertain, but recent reports have described additional general functions, including cell growth, drug resistance, and apoptosis.¹⁵ The group of protein transport includes only downregulated genes with heterogeneous functions, such as transport from the endoplasmic reticulum to the cell surface (DNAjc14) and nucleocytoplasmic shuttling (Slc4a1ap). Two genes in this category play a role in cytoskeleton remodeling, namely Ptitpnm1 (which regulates RhoA activity and cytoskleton rearrangements) and Atg4 (which cleaves the carboxyl terminal of the microtubule-associated protein light chain 3, LC3). We also found an upregulation of three genes involved in energy metabolism that may be related to increased oxygen use occurring during memory formation. In the apoptosis category, we found that Apip, a gene with antiapoptotic action, was upregulated, whereas Cyc, proapoptotic, was downregulated. Lsm7 and Lsm8 are upregulated genes included in the nucleic acid-processing category; they are involved in the stabilization of the spliceosome and function in mRNA decay.

One interesting upregulated gene is Aph1b (Presenilin stabilization factor-like), included in the protein processing category. Aph1b is part of the γ-secretase complex that displays catalytic activity toward type I transmembrane proteins. Aph1b knockdown rats show alterations in γ-secretase subunit composition that lead to an altered proteolytic processing of several γ-secretase substrates resulting in neuro-signaling pathway alterations.¹⁶

In conclusion, the major finding of the present investigation is that aged rats with good memory retention have differential expression in genes, including cytoskeleton regulation and regulatory neurosignaling pathways. Our data are partly consistent with recent reports showing a differential transcription of genes related with protein modification, transcription/translation, signal transduction, and neurotransmission in the hippocampus of aged rats showing a better performance in spatial memory tested with the Morris water maze paradigm.³ Present results may provide insight into the mechanisms underlying the maintenance of long-term declarative memory formation in the elderly. Further studies are needed to elucidate the role and the implications of the differentially expressed genes identified in the present work and to clarify the mechanisms of selective brain function vulnerability during aging.

Footnotes

Acknowledgments

This work was supported by Italian Ministry for University and Research (MIUR) grant RBNEO18R89-002.

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