Differential effect of retinoic acid and triiodothyronine on the age-related hypo-expression of neurogranin in rat
Introduction
The roles of the retinoids, derivatives of the Vitamin A, in many physiological functions such as vision, immunity, cellular differentiation or proliferation or regulation of gene expression are at least in part well known [7], [55]. The action of Vitamin A is mediated through its active metabolite, retinoic acid (RA), by two classes of nuclear receptors: the retinoic acid receptors (RARα, β and γ) able to bind all-trans stereoisomer of retinoic acid (RA), and the retinoid X receptors (RXRα, β and γ), which bind the 9-cis stereoisomer. These receptors belong to the steroid/thyroid hormone nuclear receptor superfamily and are DNA-binding proteins [1], which, upon activation by specific retinoid ligands, induce gene transcription by interacting with distinct promoter sequences in the target genes predominantly in the form of RAR/RXR heterodimers [35], [42]. Recent research concerning the role of Vitamin A in the brain suggests that retinoid signaling plays an important role during mouse embryonic development [40], and that retinoid receptors contribute to specific functions in the adult central nervous system as well [32], [45]. The presence of the two classes of retinoid receptors in different adult mouse central nervous system areas [36] and the high level of cellular retinol and retinoic acid-binding proteins in the adult brain [61] indeed, indicate an involvement of retinoids in specific physiological brain functions. In the brain, RARβ is the main isoform expressed and RXRβ and RXRγ are also largely expressed at high levels [41]. Moreover, knockout mice for RARβ and RARβ–RXRγ display an alteration of synaptic plasticity as well as substantial performance deficits in a hippocampal-dependent spatial learning task [9]. Among the genes whose expression is regulated by retinoic acid, and in addition to those coding for their own receptors, there are genes of two identified neuron specific proteins involved in synaptic plasticity: neuromodulin or GAP43 [52], [53] and neurogranin or RC3 [34]. GAP43, a pre-synaptic PKC substrate, which is associated with the cytoplasmic face of the axonal growth-cone membrane, has been implicated in different forms of synaptic plasticity, including neurite outgrowth, regeneration and long term potentiation (LTP) [2], [25]. The post-synaptic PKC substrate, RC3, is a postnatal onset protein, which accumulates in forebrain dendritic spines where a high degree of plasticity is maintained throughout life [26]. An increased amount of the phosphorylated form of RC3 has been reported during the maintenance phase of LTP [8]. More recently, it has been shown that mice lacking the RC3 gene exhibited deficits in hippocampal synaptic plasticity and spatial learning impairment [46]. RC3 is mainly expressed in the hippocampus, the striatum and the cerebral cortex [26].
Although aging is associated with a clear decline in cognitive functions [20], [49], the structural and cellular bases of these changes remain poorly defined. Recently, evidence has been presented that aging leads to hypo-expression of retinoid signaling pathway in the brain in mice and rats. This hypo-expression may be the consequence of a decrease in retinoic acid bio-availability and is associated with an age-related reduction in neuron plasticity (characterized by hypo-expression of RC3). These studies showed that older rats and mice, which displayed lower level of brain RARβ and RXRβ/γ mRNA with respect to younger adults, also had severely and specifically impaired memory performance [16], [21]. Likewise, in the same studies, retinoic acid administration restored, to pre-senescent levels, the age-related deficits observed, confirming that a fine regulation of retinoid mediated gene expression seems fundamentally important for optimal brain function.
Data comparable to those obtained in aged animals have recently been obtained in Vitamin A deficient animals, i.e. hypo-expression of retinoid nuclear receptors and RC3 as well as memory impairment. Surprisingly, RA administration to depleted animals failed to fully restore the Vitamin A deficient-related hypo-expression of RC3, and had no effect on associated cognitive deficit [18], [22]. The hypo-activity of the retinoid pathway, induced by Vitamin A deficiency, is accompanied by a hypo-activity of T3 signaling, which becomes a limiting factor, because it impedes RA from exerting its modulator effect [32].
Numerous studies have reported the close relationship between retinoid and thyroid signaling [10], [54], [58]. Like retinoic acid, triiodothyronine (T3) mediates its effect through binding to nuclear receptors (TR), which are ligand-inducible transcription factors. RXR is the common partner for the formation of RAR, or TR functional heterodimers, indicating that the retinoid and thyroid signaling pathways converge through the direct interaction of their respective nuclear receptors [38], [60]. Interestingly, the RC3 gene is well known to be at once a T3 and RA responsive gene [44].
Interestingly, alterations in thyroid function have also been frequently reported as arising with age [12], [15]. Most of the thyroid disorders described in elderly people are related to central hypothyroidism. Indeed, serum basal and free T3 concentrations have been shown to be inversely correlated with age [13], [19].
Taking these data into account, the aim of the present paper is to investigate the involvement of thyroid signaling in the function of retinoic acid and in age-related neurobiological alterations. First, the status of aged animals related to retinoid and thyroid pathways in brain was determined. Then, effects of RA or T3 administration on RA and T3 nuclear receptor expression were compared in the whole brain of aged rats. Finally, in order to fully understand the consequences on the brain functioning, we studied the expression of neuromodulin (GAP43) and neurogranin (RC3). In the whole brain, the levels of mRNA and proteins were evaluated by using respectively real time PCR and western blot analysis. On the other hand, RC3 mRNA were also quantified by an in situ hybridization study in three regions of brain enriched in RC3: hippocampus, striatum and cerebral cortex [34].
Section snippets
Experimental design
The study was conducted in accordance with the European Community's Council Directives (861609/EEC). All the experiments conformed with the Guidelines on the Handling and Training of Laboratory Animals. Weanling male Wistar rats were purchased from Harlan (Gannat, France). They were maintained in a room with a constant airflow system, controlled temperature (21–23 °C) and a 12 h light: dark cycle. The rats had ad libitum access to food and tap water. They were randomly divided into two groups
Serum retinol and triiodothyronine concentrations
A significant reduction (−20%) in serum retinol concentration was observed in aged animals relative to controls (0.91 ± 0.06 μmol/L versus 1.16 ± 0.06 μmol/L). The concentration of triiodothyronine also significantly decreased with aging even if this reduction was less than for retinol (1.11 ± 0.02 nmol/L versus 1.00 ± 0.02 nmol/L).
Expression of retinoic acid and triiodothyronine nuclear receptors and target genes (GAP43 and RC3) in the whole brain
The results are summarized in Table 1.
A significant decrease in the level of retinoic acid nuclear receptors was observed in the brain of aged animals. The amount of
Status of aged rats
In order to study the effect of T3 on brain retinoic acid and triiodothyronine signaling in aged rats, the function of these pathways was first examined in 24 months old rats. Together, all the results obtained in this experiment established the decline in the brain of retinoid but also thyroid signaling with aging. Several authors have evoked similar results in rats and mice [16], [17]. Further data shown that knockout mice for RARβ or RARβ/RXRγ exhibited a long term potentiation (LTP) deficit
Acknowledgements
This research was supported by the Institut National de la Recherche Agronomique (INRA) and by the Conseil Régional d’Aquitaine. The authors wish to thank L. Caune for animal care.
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