Effects of hCG on reduced numbers of hCG receptors in the prefrontal cortex and cerebellum of rat models of Alzheimer’s disease

ABSTRACT

Age-associated changes in the levels of luteinizing hormone and human chorionic gonadotropin (hCG) are potential risk factors for Alzheimer’s disease (AD); hCG concentration is related to the incidence of AD. The highest density of hCG receptors is in zones of the brain that are vulnerable to AD and streptozotocin (STZ) can decrease the density of this receptor. We investigated the effects of different doses of hCG on hCG receptor density in the prefrontal cortex and cerebellum in a rat model of STZ-induced AD. AD was induced by intracerebroventricular injection of 3 mg/kg STZ. The resulting AD rats were treated for 3 days with 50, 100 or 200 IU/200 μl hCG, or with saline as a control. Sections of prefrontal cortex and cerebellum were stained immunohistochemically and hCG receptor-immunoreactive (ir) neurons were counted. STZ injected into the lateral ventricles of rat brains reduced the density of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. hCG administration resulted in a significant dose-dependent increase in the number of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. The maximum increase in the number of receptors occurred following the 200 IU dose of hCG. Administration of hCG ameliorated the lowered density of hCG receptor-ir neurons in the cerebellum and prefrontal cortex in STZ-induced AD rats.     

Dual effect of beta-amyloid on α7 and α4β2 nicotinic receptors controlling the release of glutamate, aspartate and GABA in rat hippocampus

Background

We previously showed that beta-amyloid (Aβ), a peptide considered as relevant to Alzheimer''s Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer''s Disease pathology, evaluating the effect of Aβ (at different concentrations) on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4β2 and α7 subtypes). Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA.

Methodology/Findings

We used a dual approach: in vivo experiments (microdialysis technique on freely moving rats) in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus). Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aβ considered (10 µM in vivo and 100 nM in vitro). In vivo administration of 100 nM Aβ (the lowest concentration considered) potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aβ and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4β2 nAChRs selective agonists, respectively) elicited the hippocampal release of aspartate, glutamate, and GABA. High Aβ concentrations (100 nM) inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aβ concentrations (1 nM and 100 pM) selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA.

Conclusions/Significance

The results reinforce the concept that Aβ has relevant neuromodulatory effects, which may span from facilitation to inhibition of stimulated release depending upon the concentration used.     

Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1

The cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluRla cDNA and with its truncated variant which encodes the receptor termed mGluRlT (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists. This effect was completely dependent on the presence of extracellular calcium, and unlike the agonist-stimulated IP formation it was insensitive to pertussis toxin. The prolonged activation of IP formation might affect the cell physiology. In an attempt to obtain stably transfected cells, we transfected about 1.5 × 10⁶ HEK-293 cells with the plasmide conveying the full-length mGluR1a cDNA and the neomicin-resistance gene. Only 12 clones survived the antibiotic selection, and only one of these 12 clones continued to divide. The size of mRNA from the clone was smaller than the full-length mGluR1a mRNA. The shortened mRNA, revealed in the clone, apparently encoded a functional mGluR that was sensitive to glutamate, but unlike the mGluR1a, it did not respond to 1S,3R-ACPD (1S,3R-aminocyclopentane-1,3-dicarboxylic acid). A prudent use of the heterologous cell transfection technique is necessary in studying the xfunction and the pharmacology of mGluRs.     

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca(2+)]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na(+)/Ca(2+) exchanger blocker CGP37157. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca(2+) entry and PKC activity.     

The taste of monosodium glutamate (MSG), L-aspartic acid, and N-methyl-D-aspartate (NMDA) in rats: are NMDA receptors involved in MSG taste?

Monosodium glutamate (MSG) is believed to elicit a unique taste perception known as umami. We have used conditioned taste aversion assays in rats to compare taste responses elicited by the glutamate receptor agonists MSG, L-aspartic acid (L-Asp), and N-methyl-D-aspartate (NMDA), and to determine if these compounds share a common taste quality. This information could shed new light upon the receptor mechanisms of glutamate taste transduction. Taste aversions to either MSG, L-Asp or NMDA were produced by injecting rats with LiCl after they had ingested one of these stimuli. Subsequently, rats were tested to determine whether they would ingest any of the above compounds. The results clearly show that a conditioned aversion to MSG generalized to L-Asp in a dose-dependent manner. Conversely, rats conditioned to avoid L-Asp also avoided MSG. Conditioned aversions to MSG or L-Asp generalized to sucrose when amiloride was included in all solutions. Importantly, aversions to MSG or L-Asp did not generalize to NMDA, NaCl or KCl, and aversions to NMDA did not generalize to MSG, L-Asp, sucrose or KCl. These data indicate that rats perceive MSG and L-Asp as similar tastes, whereas NMDA, NaCl and KCl elicit other tastes. The results do not support a dominant role for the NMDA subtype of glutamate receptors in taste transduction for MSG (i.e. umami) in rats.     

Expression of estrogen receptor (ER) -α and -βtranscripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb

INTRODUCTIONEstrogen has been known to exert extensive effects via estrogen receptor (ER) on diverse physio-logical and develoPmental functions of the brain[1,2]. It has been observed that the distribution ofthe classical ER subtype-a (ERa) and the recentlycharacterized novel ER subtype--fi (ERg), and theirexpression patterns (ERcr/ERfi) vary greatly amongvarious brain regions[1, 3]. These evidences suggestthat each ER subtype may play a different role inestrogen's effects on the br…     

Cyclosporine therapy of rat heart allograft recipients and release of interleukins (IL 1, IL 2, IL 3): a role for IL 3 in graft tolerance?

LEW rat recipients of (LEW X BN)F1 strain heterotopic cardiac transplants treated with cyclosporine A (CsA) (15 mg/kg/day intramuscularly, 7 days) retain grafts indefinitely despite drug withdrawal. Donor-specific suppressor T cells that are active in passive transfer experiments have been harvested from long-term CsA-treated hosts. Although CsA is known to inhibit in vitro cytokine release, the in vivo effects of the CsA on the lymphokine cascade are not known. We investigated the action of the drug upon spontaneous and mitogen-induced interleukin 1 (IL 1), interleukin 2 (IL 2), and interleukin 3 (IL 3) release by spleen cells obtained from the following groups of rats: 1) normal, i.e., untreated and ungrafted; 2) grafted, acutely rejecting; 3) grafted, actively treated; and 4) under CsA-induced state of "tolerance." The results demonstrate that in vivo CsA therapy inhibits monocyte (IL 1 release) as well as lymphocyte function (IL 2 and IL 3 release) only during the inductive phase (the 7 days of treatment). During the "tolerant" phase, mitogen (Con A and LPS)-induced release of interleukins was quantitatively similar to that noted in normal animals. In contrast, a remarkable increase in the spontaneous production of IL 3 was observed in the "tolerant" group. Because cytokine release is not inhibited in the "tolerant" state, our data strongly support the concept that maintenance of the state of unresponsiveness is governed by the emergence of suppressor cells. The correlation of increased spontaneous production of IL 3 during this period leads us to postulate that this interleukin may be implicated in the activation or clonal expansion of suppressor cells, and hence may play a role in graft tolerance.     

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling,down-regulation of PPARγ and induction of iNOS

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC₅₀ of 10 μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.     

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric α7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via α7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.