Adult-onset hypothyroidism induces the amyloidogenic pathway of amyloid precursor protein processing in the rat hippocampus.

Thyroid dysfunction and dementia are conditions that become more prevalent with advancing age. Localised hypothyroidism of the central nervous system has been sugested in some patients with Alzheimer's disease. We investigated the consequence of adult-onset hypothyroidism on beta-amyloid precursor protein (APP) degrading pathways in rats treated with propylthiouracyl over a period of 5 weeks. We evaluated the amount of 3,5,3'-triiodothyronine nuclear receptors (TRalpha1 and TRbeta) and the expression of some APP processing indicators (i.e. APP, ADAM 10, BACE and PS1). The activity of secretases and Abeta peptides has been also quantified. The results obtained show that hypoactivity of the thyroid signalling pathway in the hippocampus induced an increase in the APP770-751/APP695 ratio accompanied by a modification in the amyloidogenic pathway for APP processing, leading to an increased amount of Abeta peptides. In this area of the brain, modification in the non-amyloidogenic pathway of APP processing characterised by alpha-secretase activity was only approximately 10% in hypothyroid rats compared to control rats. We suggest that hypothyroidism, which becomes more prevalent with advancing age, increased the vulnerability to the formation of amyloid deposits.

Thyroid hormone receptor alpha plays an essential role in the normalisation of adult-onset hypothyroidism-related hypoexpression of synaptic plasticity target genes in striatum.

Thyroid hormone (TH) deficiency leads to molecular changes resulting in behavioural deficits. TH action is mediated by two types of nuclear receptors (TRs), TRalpha and TRbeta, which control target gene transcription. The relative contributions of the two TR products in mediating adult TH responses are poorly understood. As TRalpha1 transcripts are widely distributed in the brain, they presumably mediate most of the TH effects. This report examines the role and specific functions of T3 receptor isoforms on regulation of striatal synaptic plasticity indicators using adult hypothyroid mutant mice that fail to express single or multiple TR gene products. We then evaluated the effect of this hypothyroidism, with or without subsequent administration of T3, on T3 nuclear receptor (TRalpha1, TRbeta) and synaptic plasticity gene expression in TRalpha(0/0), TRbeta(-/-) and wild-type 129/SV mice. Hypothyroid wild-type mice exhibited reduced TRbeta, RC3, CaMKII and Rhes expression. The mRNA levels of Rhes and CaMKII were the same in all three hypothyroid substrains. By contrast, hypothyroid TRbeta(-/-) mice had higher RC3 mRNA levels than wild-type. T3 administration restored TRbeta, RC3 and CaMKII levels in hypothyroid wild-type mice, without significant Rhes upregulation. T3 administration normalised expression of all genes studied in hypothyroid TRbeta(-/-) but not TRalpha(0/0) mice. Thus, TRalpha apparently plays an essential role in restoring the expression of the TH-regulated genes potentially involved in striatal synaptic plasticity.

Decreased expression of retinoid nuclear receptor (RAR alpha and RAR gamma) mRNA determined by real-time quantitative RT-PCR in peripheral blood mononuclear cells of hypothyroid patients.

In vivo assessment of the cellular impact of thyroid hormones on target tissues might be of help for physiological studies and to evaluate the consequences of various diseases of the thyroid gland in humans. Given the tenuous relationship between retinoid and tri-iodothyronine (T3) status and that retinoids have also intracellular roles, the aim of this study was to determine the effect of hypothyroidism on the expression of T3 nuclear receptors (TR) and retinoic acid nuclear receptors (RAR, RXR) in human peripheral blood mononuclear cells (PBMC). Using real time RT-PCR, we quantified the relative amount of mRNA of the thyroid (TR alpha and TR beta) and retinoid (RAR alpha, RAR gamma, and RXR alpha) nuclear receptors in PBMC of euthyroid (n = 22) compared with hypothyroid (n = 22) subjects. Classical plasma parameters (free T3 (FT3), free thyroxine (T4) (FT4), thyroid-stimulating hormone (TSH), retinol (ROH), retinol-binding protein (RBP) and transthyretin (TTR)) were also measured. In hypothyroid subjects, the concentration of TSH was elevated, and dramatically low T3 and T4 concentrations were associated with a decrease in the expression of TR beta. Expression of RAR alpha and RAR gamma significantly decreased in hypothyroid versus control subjects, while an increased concentration of ROH was emphasised by hypothyroidism. These results first indicated that primary hypothyroidism induces hypoactivation of the retinoid nuclear pathway in PBMC, which was not predicted by the plasma ROH level. Further investigations will be necessary to evaluate these parameters in very small changes in thyroid hormone production such as mild (subclinical) hypothyroidism.

Differential effect of retinoic acid and triiodothyronine on the age-related hypo-expression of neurogranin in rat.

Given the important role of retinoids and thyroid hormone for optimal brain functioning and the tenuous relationship between retinoic acid (RA) and triiodothyronine (T3) signalings, we compared the effects of RA or T3 administrations on RA and T3 nuclear receptors (RAR, RXR and TR) and on their target genes, neuromodulin (GAP43) and neurogranin (RC3) in 24-month-old rats. Quantitative real time PCR and western blot analysis allowed us to verify that retinoid and thyroid signalings and GAP43 and RC3 expression are affected by age. By in situ hybridization we observed a decreased expression of RC3 in hippocampus, striatum and cerebral cortex. RARbeta, RXRbeta/gamma and GAP43 were up-regulated by RA as well as T3 treatment. The abundance of TRalpha/beta mRNA and RC3 expression were only increased by T3 administration in the whole brain. This up-regulator effect of T3 on RC3 was only observed in the striatum. During aging, T3 become a limiting factor alone able to correct the age-related concomitant hypo-activation of retinoid and thyroid signalings and alterations of synaptic plasticity.

Expression of neurogranin and neuromodulin is affected in the striatum of vitamin A-deprived rats.

Our previous data showed that vitamin A deficiency (VAD) induces, in whole brain, a reduced amount of mRNA for brain retinoic acid (RA) and triiodothyronine (T3) nuclear receptors (i.e., RAR, RXR, and TR, respectively), which is accompanied by reduced amounts of mRNA and protein of neurogranin (RC3, a neuronal protein involved in synaptic plasticity) as well as selective behavioral impairment. Given the important role of retinoids for optimal brain functioning, the effects of vitamin A depletion and subsequent administration of RA or T3 on the mRNA levels of RA and T3 nuclear receptors and on two target genes' (RC3 and neuromodulin or GAP43) mRNA and protein levels were examined in the hippocampus, striatum, and cerebral cortex. A quantitative real-time polymerase chain reaction (PCR), in situ hybridization, and Western blot analysis demonstrated that the striatal region is the brain site where both RA and T3 signaling pathways are most affected by VAD. Indeed, rats fed a vitamin A-free diet for 10 weeks exhibited decreased expression of RAR, RXR, TR, RC3, and GAP43 in the striatum. The administration of T3 to these vitamin A-deprived rats reversed the reduction in mRNA levels of RA and T3 nuclear receptors and in mRNA and protein levels of target genes in this region. These data suggest that modifications that appear preferentially in the striatum, a region highly sensitive to vitamin A bioavailability, may contribute to neurobiological alterations and the spatial learning impairment that occurs in vitamin A-deprived animals.

Retinoic acid reverses the PTU related decrease in neurogranin level in mice brain.

Recent data have shown that fine regulation of retinoid mediated gene expression is fundamentally important for optimal brain functioning in aged mice. Nevertheless, alteration of the thyroid hormone signalling pathway may be a limiting factor, which impedes retinoic acid (RA) from exerting its modulating effect. Mild hypothyroidism is often described in the elderly. Thus, in the present study, it was of interest to determine if RA exerts its neurological modulating effect in mild hypothyroidism. To obtain further insight into this question, mice were submitted to a low propylthiouracyl (PTU) drink (0.05%) in order to slightly reduce the serum level of triiodothyronine (T3). A quantitative evaluation of RA nuclear receptors (RAR, RXR), T3 nuclear receptor (TR) and of neurogranin (RC3, a RA target gene which codes for a protein considered as a good marker of synaptic plasticity) in PTU treated mice injected with vehicle or RA or T3 was carried out. The PTU-related decrease in expression of RAR, RXR and RC3 was restored following RA or T3 administration, as observed in aged mice. The amount of TR mRNA, which was not affected in PTU treated mice, was increased only after T3 treatment as observed in overt hypothyroidism. These results suggest that neurobiological alterations observed in aged mice are probably related to RA and T3 signalling pathway modifications associated, in part, with mild changes in thyroid function.

A retinoic acid receptor antagonist suppresses brain retinoic acid receptor overexpression and reverses a working memory deficit induced by chronic ethanol consumption in mice.

BACKGROUND: Chronic ethanol consumption induces disorders in the biosynthesis of retinoic acid, an active derivative of vitamin A. Recent evidence suggests that an alteration in the retinoic acid signaling pathway leads to impairments in learning and memory in adult mice. We have previously shown that chronic ethanol consumption in mice produces an increased expression of the brain retinoic acid receptor beta (RARbeta) mRNA. These results prompted us to examine whether suppressing the overexpression of retinoid receptors in alcohol-treated mice by RAR antagonist administration would reverse their cognitive impairment. METHODS: After 10 months of ethanol consumption (12% v/v in drinking water), C57BL/6 mice were submitted to a working memory task in a T-maze. Then, mice of the control and the ethanol-treated groups received an RARbeta antagonist (CD2665 0.6 mg/kg) for 22 days. The behavioral effect of CD2665 administration was evaluated on a spontaneous alternation task and the neurochemical effect was measured by quantifying the mRNA expression of RARalpha, RARbeta, retinoid X receptor (RXRbeta/gamma) and tissue transglutaminase (tTG; a retinoic acid-target gene). RESULTS: Mice submitted to ethanol treatment exhibited a progressive decrease in spontaneous alternation rates over successive trials. Moreover, these mice displayed an increased expression of brain RARbeta and RXRbeta/gamma mRNA, together with an increased level of tTG mRNA and enzymatic activity. The administration of CD2665 to alcohol-treated mice totally reversed the working memory deficit and suppressed the overexpression of brain RARbeta, RXRbeta/gamma and tTG mRNA, whereas the same treatment in control mice decreased only the RARbeta mRNA level without affecting memory performance. CONCLUSION: These data point to the potential role of the retinoid signaling pathway in memory processes and suggest that the overexpression of brain RARbeta and RXRbeta/gamma could be responsible, at least in part, for some memory impairments observed during chronic ethanol consumption.

Selective age-related changes in the PKC-sensitive, calmodulin-binding protein, neurogranin, in the mouse brain.

Brain ageing is associated with a dysregulation of intracellular calcium (Ca(2+)) homeostasis which leads to deficits in Ca(2+)-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca(2+) and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca(2+)/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.

Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling.

Vitamin A and its derivatives, the retinoids, have been implicated recently in the synaptic plasticity of the hippocampus and might therefore play a role in associated cognitive functions. Acting via transcription factors, retinoids can regulate gene expression via their nuclear receptors [retinoic acid receptors (RARs) and retinoid X receptors]. In a series of experiments, the present study investigated the possible role of age-related downregulation of retinoid-mediated transcription events in the cognitive decline seen in aged mice. We observed that the brain (and hippocampal) levels of retinoid receptors and the expression of specific associated target genes were restored to presenescent (adult) levels in aged mice after acute administration (150 microg/kg, s.c.) of retinoic acid (RA). These effects of RA, however, could be abolished by the coadministration of an RAR antagonist. RA was also demonstrated to alleviate the age-related deficit in the CA1 long-term potentiation efficacy of aged mice in vivo. Moreover, RA was found to alleviate completely the performance deficit of aged mice to the control level in a two-stage spatial discrimination paradigm designed to assess relational memory. This promnesic effect of RA was again susceptible to abolition by RAR antagonist treatment. The parallel molecular, cellular, and behavioral correlates associated with the decrease of retinoid receptor expression and its normalization demonstrated here suggest that the fine regulation of retinoid-mediated gene expression is fundamentally important to optimal brain functioning and higher cognition. Specifically, a naturally occurring dysregulation of retinoid-mediated molecular events might be a potential etiological factor for cognitive deterioration during senescence.

Vitamin A Deficiency Decreases the Expression of RARß and RXRß/γ in Adult Mouse Brain: Effect of RA Administration.

Recent studies have revealed a novel and unexpected role of vitamin A, via its nuclear receptors, in higher cognitive functions. We examined, in mouse brain, the impact of vitamin A status on the level of retinoic acid nuclear receptor (RAR and RXR) expression and on one of their responsive genes, that of the tissue transglutaminase (tTG). Weanling male C57B1/6 mice fed a vitamin A deficient diet developed a vitamin A deficiency which was characterized, after 26 weeks, by the depletion of serum retinol, liver retinol and retinyl palmitate, and by the decreased activity of liver tTG. After 27 weeks of the diet, the vitamin A depleted mice exhibited a significantly lower amount of brain RAR ß and RXR ß/γ mRNA relative to control mice. Vitamin A deficiency also resulted in a reduced expression of tTG. When 35-week depleted mice were subsequently administrated RA for 28 days, the expression of RA nuclear receptors and tTG was significantly induced. The vitamin A deficiency mouse model and repletion provides a physiological system for monitoring the effects of vitamin A status on gene expression and on neurobiological processes in the adult brain.

Aging decreases the abundance of retinoic acid (RAR) and triiodothyronine (TR) nuclear receptor mRNA in rat brain: effect of the administration of retinoids.

Aging is accompanied by troubles resulting from changes in hormonal and nutritional status. Therefore, the abundance of mRNA coding for triiodothyronine (TR) and retinoic acid (RA) nuclear receptors was studied in the brain of young, adult and aged (2.5, 6 and 24 months, respectively) rats. In the brain of aged rats, there was a lower abundance of TR and RAR mRNA and a lower activity of tissue transglutaminase (tTG), an enzyme the gene of which is a target for retinoids. Administration of RA in these rats restored TR and RAR mRNA and the activity of tTG in the brain. The importance of these observations to the function of the aged brain is discussed.

Age-related decreases in mRNA for brain nuclear receptors and target genes are reversed by retinoic acid treatment.

Ageing is accompanied by certain problems resulting from changes of hormonal status, in particular thyroid hormone (T3) status and vitamin A status. Since retinoic acid (RA), the active metabolite of vitamin A, and T3 play physiological roles in the adult brain, the effect of ageing on the amounts of mRNA for retinoic acid (RAR and RXR) and triiodothyronine (TR) nuclear receptors were studied. Also, the expression of RA and T3 target genes, tissue transglutaminase (tTG) and neurogranin (RC3), was measured in the whole brain and in the hippocampus of mice. Relative to young (3 months) mice, aged (22 months) mice exhibited lower amounts of RAR, RXR and TR mRNA concomitantly with a lower expression of tTG and RC3. RA administration to old mice (24 h before sacrifice) was able to restore the amount of mRNA of nuclear receptors and of RC3. It is hypothesized that a decrease in the cellular action of RA and T3 could play a role, via a decrease in the expression of RC3, in the alteration of synaptic plasticity occurring in aged mice.

Aging decreases retinoic acid and triiodothyronine nuclear expression in rat liver: exogenous retinol and retinoic acid differentially modulate this decreased expression.

The expression of nuclear receptors of retinoic acid (RAR) and triiodothyronine (TR) was analyzed in the liver of rats aged 2.5 (young), 6 (adult) and 24 (aged) months. In aged rats, decreased binding properties, binding capacity (Cmax) and affinity (Ka), of nuclear receptors were observed. This resulted, at least in part, from decreased transcription of receptor genes in that the amount of their mRNA also decreased. Moreover, the activity of malic enzyme (ME) and tissue transglutaminase (tTG), whose genes are TR and RAR responsive, respectively, was reduced in aged rats. These results are in agreement with the decreased binding capacity of these receptors. An inducer-related increase of RAR and TR expression was observed 24 h after a single dose of retinoic acid administration (5 mg/kg), while retinol administration (retinyl palmitate, 13 mg/kg) was without incidence on nuclear receptor expression in aged rats.