Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: A first study

Figures & data

Table I.  Changes in plasma corticosterone, body weight and protein expression.

	Sham-treated (n = 7)	Corticosterone (n = 7)	Statistical significance
Plasma corticosterone (μg/100 ml)	20.1 ± 8.4	33.8 ± 7.6	*
Body weight (g)	556 ± 44	494 ± 31	*
Total hippocampal protein (μg/ml)	33.2 ± 9.4	30.9 ± 3.2	n.s.
Relative PEBP1 expression (a.u.)	1.0 ± 0.89	0.58 ± 0.52	*

Corticosterone was quantified in plasma using a specific RIA. Data are given as means ± SD for n = 7 animals in each group after 60 days of treatment. Rats had initial mean body weight of 576 ± 64 g. PEBP1 expression was measured by quantitative western blotting and data were normalized to sham-treated mean. *P < 0.05, one-way ANOVA, post hoc Tukey test; n.s., not significant; a.u., arbitrary units.

Figure 1 Decreasing memory performance after chronic corticosterone treatment for 60 days. Whereas no difference between rats is seen at the start of the experiment, both working memory (number of food-rewarded visits/number of visits and revisits to the baited set of holes in % with SD) and reference memory (number of visits and revisits to the baited set of holes/number of visits and revisits to all holes in % with SD) performances decrease after 60 days of corticosterone treatment (n = 7; *, P < 0.05). Values are mean ± SD.

Figure 2 Differential expression of hippocampal proteins after chronic corticosterone. (A) The 2D gel electropherogram shows both spots of the corticosterone group (green) and the sham-treated group (purple) in false-color coding. Overlaid spots appear white. The position of phosphatidylethanolamine binding protein (PEBP1, MW approx. 21 kDa) is indicated by an arrow. (B) Three-dimensional reconstruction of the PEBP1 expression data from the 2D gels show decreased PEBP1 expression after 60 days of corticosterone treatment to roughly half (46%) as compared to the sham-treated group.

Table II.  Differentially expressed proteins in the rat hippocampus after chronic corticosterone treatment.

Spot no.	Accession no.	Protein name	Focus protein identity	Mean sham (n = 3)	SD sham	Mean cst (n = 3)	SD cst	P	IF (corticosterone/sham-treated)	Mascot score	Theoretical pI	Theoretical MW (Da)
		Upregulated
241	gi|51859516	Heat shock 90 kDa protein 1, beta	INA	3.72	1.12	21.39	9.12	0.003	5.75	122	4.7	83,631
416	gi|9506811	Internexin, alpha	INA	14.32	3.63	43.97	18.25	0.012	3.07	259	5.0	56,253
676	gi|16757986	Phosphoglycerate kinase 1		12.68	3.98	34.85	13.21	0.012	2.75	*	7.5	44,423
689	gi|91997	Aspartate transaminase  (EC 2.6.1.1), cytosolic		6.31	2.86	16.26	6.29	0.018	2.58	188	7.3	46,627
602	gi|31542401	Creatine kinase, brain		18.92	6.08	48.09	19.80	0.022	2.54	214	5.3	42,970
922	gi|9506445	Carbonic anhydrase 2	CA2	4.92	1.97	10.72	3.99	0.032	2.18	136	7.5	29,267
821	gi|50925725	Pyruvate dehydrogenase  (lipoamide), beta		29.49	8.46	63.69	19.81	0.012	2.16	169	6.7	39,299
520	gi|8393431	Glial fibrillary acidic protein	GFAP	15.57	4.18	32.70	12.60	0.013	2.10	283	5.2	49,970
520	gi|54792127	ATPase, H+ transport  mito F1, beta	ATP5B	15.57	4.18	32.70	12.60	0.013	2.10	81	5.0	56,318
762	gi|55778661	Sirtuin 2		4.44	1.31	9.25	3.10	0.022	2.08	130	7.1	39,921
330	gi|1000439	Glucose-related protein grp75		11.41	4.39	23.31	7.22	0.023	2.04	210	6.1	73,984
654	gi|6978431	Acyl coenzyme A dehydrogenase,  long chain		8.53	3.41	17.17	6.44	0.047	2.01	*	7.6	47,872
610	gi|31542401	Creatine kinase, brain		32.14	11.44	64.36	17.11	0.013	2.00	176	5.3	42,970
436	gi|11560024	Heat shock protein 1  (chaperonin)	HSPD1	31.94	9.51	58.53	16.24	0.023	1.83	218	6.0	61,098
759	gi|13591894	Aldo-keto reductase family 1,  memberA1	AKR1A1	10.95	2.44	17.42	5.36	0.034	1.59	157	7.4	36,711
682	gi|8393349	Fumarylacetoacetate hydrolase		7.70	2.48	12.15	3.05	0.043	1.58	83	7.2	46,231
845	gi|34858672	Similar to RIKEN cDNA  B430104H02		8.39	2.53	13.12	4.03	0.043	1.56	105	8.3	40,884
693	gi|13928690	Isocitrate dehydrogenase 1,  soluble		8.08	2.08	12.40	2.62	0.013	1.53	*	6.5	46,734
853	gi|20913941	Pyrroline-5-carboxylase  reductase (P5CR)		7.62	2.60	11.53	3.15	0.049	1.51	*	7.7	33,673
496	gi|6679745	Fascin homolog 1 (actin bundling  protein); fascin 1	FSCN1	10.48	2.39	15.57	4.11	0.040	1.49	*	6.4	54,508
717	gi|68188	Fructose-bisphosphate aldolase  (EC 4.1.2.13) C		30.33	7.59	44.63	8.45	0.024	1.47	159	7.2	39,721
965	gi|6978873	Guanidinoacetate methyl transferase		19.27	7.11	27.84	4.35	0.037	1.44	*	5.7	26,276
709	gi|30421120	Actin monomer-binding protein  twinfilin-2		6.95	0.74	9.77	2.33	0.023	1.41	97	6.8	39,674
383	gi|2190337	Albumin		27.40	6.65	38.29	6.23	0.031	1.40	*	5.8	69,293
531	gi|38512279	ATPase, H+ transport mito F1,  alpha isoform 1	ATP5A1	12.45	3.12	16.19	2.25	0.046	1.30	129	9.7	59,831
473	gi|55391497	ATPase, H+ transporting, V1  subunit B, isoform 2	ATP6V1B2	28.79	4.23	36.51	6.65	0.045	1.27	281	6.3	15,228
569	gi|7533042	Guanine deaminase		18.46	2.96	23.33	2.29	0.012	1.26	248	5.5	51,439
656	gi|13928690	Isocitrate dehydrogenase 1  (NADP+), soluble		33.32	5.07	40.87	4.71	0.029	1.23	112	7.0	47,047
656	gi|38181948	Glutamine synthetase1		33.32	5.07	40.87	4.71	0.03	1.23	207	7.0	42,983
Downregulated
881	gi|8394060	Proteasome (prosome, macropain),  alpha type 1		7.19	1.19	5.78	0.64	0.035	− 1.25	*	6.2	29,598
727	gi|46391108	NADH dehydrogenase 1 alpha  subcomplex10		6.68	2.31	3.94	0.84	0.030	− 1.69	123	5.5	31,852
563	gi|6978809	Enolase 1		10.48	3.03	6.16	1.45	0.034	− 1.69	*	6.2	46,997
746	gi|46391108	NADH dehydrogenase 1 alpha  subcomplex 10		7.87	1.88	4.52	1.47	0.024	− 1.75	125	5.5	31,852
812	gi|13929082	Pyridoxal (pyridoxine, vitamin B6)  kinase		13.25	3.78	6.32	2.18	0.012	− 2.08	102	6.8	35,114
1048	gi|8393910	Phosphatidylethanolamine binding protein	PEBP1	3.52	1.37	1.64	0.48	0.031	− 2.17	*	5.5	20,670
385	gi|34874349	Collapsin response mediator protein 2(CRMP − 2)		5.99	1.91	2.69	0.94	0.012	− 2.22	97	6.8	82,050
623	gi|809561	Actin, gamma		9.07	3.44	3.43	1.37	0.016	− 2.63	182	5.7	41,335
623	gi|71620	actin, beta		9.07	3.44	3.43	1.37	0.016	− 2.63	180	5.2	42,066
347	gi|56385	Hsc70 ps1		37.25	10.43	13.18	4.86	0.002	− 2.86	218	5.3	71,112
1089	gi|19924089	Nucleoside diphosphate kinase		2.17	0.89	0.72	0.28	0.014	− 3.03	140	6.3	17,296
1016	gi|23821758	UMP CMP kinase, deoxycytidylate kinase		6.25	2.81	1.55	0.57	0.010	− 4.00	94	5.5	2379
697	gi|809561	Actin, gamma		38.29	14.90	7.18	2.96	0.019	− 5.26	94	5.7	41,335
697	gi|71620	Actin, beta		38.29	14.90	7.18	2.96	0.019	− 5.26	93	5.2	42,066
468	gi|38328248	Tubulin, alpha 1	TUBA3		95.61	40.63	11.89	5.02	0.020	− 8.33	122	4.8
468	gi|11693172	Calreticulin	CALR	95.61	40.63	11.89	5.02	0.020	− 8.33	97	4.1	48,137

The table lists upregulated (upper section) and downregulated (lower section) proteins in the corticosterone group (CST). Induction factors (IF) represent the relative n-fold expression of the identified candidates in the corticosterone group; negative values represent a downregulation in the corticosterone group (CST). Proteins have been identified by peptide mass fingerprinting (PMF, entries with a Mascot score) or polynomial image warping (entries with a * in the Mascot score field). GenBank accession is listed (http://www.ncbi.nlm.nih.gov/) together with isoelectric points (pI) and molecular weights (MW). Mascot-delivered probability based scores greater than 75 reflect significant database matches (P < 0.05) and are regarded as a quality parameter for correct protein identification in PMF. The abbreviation for focus proteins identifies these proteins in the functional network map (Figure 3).

Figure 3 Functional network mapping of differentially expressed hippocampal proteins after chronic corticosterone. Proteins with expression altered after 60 days of corticosterone treatment were analysed regarding their biological functional network using the IPA (Ingenuity Systems, www.ingenuity.com). The network is displayed as nodes (proteins) and lines (biological relationships between the nodes). Focus proteins which are downregulated under corticosterone treatment are shown in green, proteins upregulated under chronic corticosterone in red. Protein abbreviations can be inferred from Table II. The network contains the phosphatidylethanolamine binding protein-1, PEBP1, and other putative chronic corticosterone target proteins such as calreticulin, CALR, various components associated with synaptic plasticity (internexin alpha, INA; fascin 1, FSCN1; tubulin alpha, TUBA3; glial acidic fibrillary protein, GFAP), as well as metabolic factors (carbonic anhydrase 2, CA2; aldo-keto reductase family 1 member A1, AKR1A1; mitochondrial H⁺-transporting ATPase F1 alpha isoform 1, ATP5A1, and beta isoform, ATP5B, as well as H⁺-transporting ATPase V1 subunit B isoform 2, ATP6V1B2) and chaperones (heat shock protein 1, HSPD1).

Figure 4 Western blot analysis of PEBP1 expression in the rat hippocampus after 60 days of corticosterone treatment. (A) The panel shows the relative quantification of PEBP1 expression in sham- and corticosterone-treated rats in arbitrary units (a.u.; n = 7). Bars above the mean show the standard deviation. Of note, the largest PEBP1 expression value in the corticosterone group is only slightly greater than the smallest expression value in the sham-treated group. (B) Original gel image obtained by western blotting of the lysates and respective levels of a housekeeping protein. A single PEBP1 band is located at about 22 kDa.

Figure 5 Model of PEBP1 as a mediator in adaptive chronic glucocorticoid-induced response of the brain. Via long-term (acetylcholine) system fatigue upon chronic corticosterone impact, a septo-hippocampal system (SHS) negative override can occur. This results in impaired function over time (right branch). Its feed-forward contributes to the inhibition of several transmitter systems in the hippocampus (black box) leading to circuit defects and reduction of hippocampus-sensitive memory capacities. An alternative transformation function may include regulation of PEBP1 (HCNP) expression, possibly acting together with stem cell proliferation and neurogenesis (NG) to influence circuit integrity. In parallel, PEBP1 inhibition may result from glutamate excitotoxicity and subsequent effects of reactive oxygen species (ROS) (middle branch). Likely, PEBP1 expression is regulated on the levels of the genome, proteome and physiome, i.e. integrated biological function, and makes up only one component of a dynamic response variability that includes manifold interactions with different stages of response to chronic corticosterone (or stress). Question marks indicate unclear relations,+/ − signs stimulatory/inhibitory effects.