Mariann Blum

Central nicotinic receptors, neurotrophic factors and neuroprotection

The multiple combinations of nAChR subunits identified in central nervous structures possess distinct pharmacological and physiological properties. A growing number of data have shown that compounds interacting with neuronal nAChRs have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improving of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimers and Parkinsons disease. Taken together experimental and clinical data largely indicate a neuroprotective/trophic role of nAChR activation involving mainly alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR antagonists, and by potent nAChR agonists recently found displaying efficacy and/or larger selective affinities than (-)-nicotine for neuronal nAChR subtypes. A neurotrophic factor gene regulation by nAChR signalling has been taken into consideration as possible mechanism involved in neuroprotective/trophic effects by nAChR activation and has evidenced an involvement of the fibroblast growth factor (FGF-2) gene as a target of nAChR signalling. These findings suggested that FGF-2 could be involved, according to the FGF-2 neurotrophic functions, in nAChR mechanisms mediating the neuronal survival, trophism and plasticity.

Induction of Interleukin-1 Associated with Compensatory Dopaminergic Sprouting in the Denervated Striatum of Young Mice: Model of Aging and Neurodegenerative Disease

Young mice challenged with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively destroys the substantia nigra dopaminergic neurons in the midbrain, exhibit spontaneous recovery of dopaminergic nerve terminals. However, such recovery becomes attenuated with age. Here we report that newly sprouted fibers originate from spared dopaminergic neurons in the ventral tegmental area. We found that interleukin-1 (IL-1), an immune response-generated cytokine that can enhance dopaminergic sprouting when exogenously applied, increased dramatically in the denervated striatum of young mice (2 months) compared with middle-aged mice (8 months) after MPTP treatment. Young mice displayed a maximal 500% induction of IL-1α synthesis that remained elevated for several weeks in the dorsal and ventral striatum, whereas middle-aged mice exhibited a modest 135% induction exclusively in the dorsal striatum for a week. IL-1α immunoreactivity was localized in GFAP-immunoreactive hypertrophied astrocytes and neurons within the denervated striatum of young mice. However, no induction of IL-1α mRNA was seen in the midbrain in either age group despite glial activation. Because we have reported that IL-1 can regulate astroglia-derived dopaminergic neurotrophic factors, it was surprising that no changes were observed in acidic and basic fibroblast growth factor or glial cell line-derived neurotrophic factor mRNA levels associated with MPTP-induced plasticity of dopaminergic neurons in the striatum of young mice. Interestingly, we found that dopaminergic neurons express IL-1 receptors, thus suggesting that IL-1α could directly act as a target-derived dopaminergic neurotrophic factor to initiate or enhance the sprouting of dopaminergic axonal terminals. These findings strongly suggest that IL-1α could play an important role in MPTP-induced plasticity of dopaminergic neurons.

A null mutation in TGF-α leads to a reduction in midbrain dopaminergic neurons in the substantia nigra

Transforming growth factor (TGF)-α is neurotrophic for midbrain dopaminergic neurons in vitro. Here I investigated whether a null mutation in the TGF-α gene affects the normal development or survival of dopaminergic neurons in either the substantial nigra (SN) or the ventral tegmental area (VTA). The SN of TGF-α knockout mice contained 50% fewer dopaminergic neurons than the control SN, but VTA neuron number was unchanged. In addition, the overall volume of the dorsal striatum was reduced by 20%. Newborn mice showed a similar decrease in the number of SN dopaminergic neurons, suggesting that TGF-α is unlikely to regulate developmental neuron death. These studies indicate that TGF-α is required for the normal proliferation or differentiation of a select population of dopaminergic neurons within the SN.

Differential response of ventral midbrain and striatal progenitor cells to lesions of the nigrostriatal dopaminergic projection.

In response to injury, progenitor cells in the adult brain can proliferate and generate new neurons and/or glia, which may then participate in injury-induced compensatory processes. In this study, we explore the ability of young adult mice to generate new cells in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesions, which selectively kill nigrostriatal dopaminergic neurons. Using the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU), we labeled dividing cells 3, 10, and 15 days after MPTP lesion. A robust proliferative response was seen specifically in the substantia nigra (SN) and the dorsal striatum 3 days postlesion; the response persisted 10–14 days. To explore the fate of proliferative cells, we administered BrdU 3 days postlesion and examined the phenotype of BrdU+ cells at various times thereafter, using double immunolabeling. In the striatum, nearly all newly-generated cells rapidly differentiated into GFAP+ astrocytes that participated in the injury-induced glial reaction. In the SN, however, reactive astroglia were not BrdU+. Some midbrain cells co-immunostained for BrdU and Mac-1, a microglial marker. However, most BrdU+ cells in the SN failed to express markers for microglia, astroglia, oligodendroglia, or neurons, suggesting that they may remain as uncommitted progenitors. Thus, progenitors in the vicinity of the degenerating dopaminergic cell bodies respond differently to lesion than progenitors in the vicinity of the degenerating axon terminal. Although the putative midbrain progenitors appear uncommitted 22 days after their birth, it is possible that they may adopt neural or glial fates if allowed to survive longer, or if exposed to exogenous factors.

Differential Expression of ErbB3 and ErbB4 Neuregulin Receptors in Dopamine Neurons and Forebrain Areas of the Adult Rat

Neuregulins have been shown to play an important role in the development of the central nervous system, but their function in adult tissues is still unclear. We investigated the expression of the neuregulin receptors erbB3 and erbB4 in the adult rat brain by in situ hybridization histochemistry. Areas with considerable expression of erbB4 receptor mRNA include cortex, amygdala, hippocampus, medial habenula, reticular thalamic nucleus, several hypothalamic nuclei, subthalamic nucleus, substantia nigra pars compacta, and ventral tegmental area. Immunostaining for tyrosine hydroxylase and dopamine depletion by 6-hydroxydopamine indicate that erbB4 is expressed in dopamine neurons in the latter two nuclei. Substantial erbB4 expression is also present in clusters of cells along the ventral and medial border of the striatum/nucleus accumbens and in the subependymal zone along the lateral and olfactory ventricles (rostral migratory stream), suggesting a role for neuregulins in adult cell proliferation. In contrast, erbB3 mRNA is mostly expressed in white matter throughout the brain and in the ependyma of the ventral half of the third ventricle (tanycytes). These results demonstrate that expression of erbB3 and erbB4 receptors is widespread in the adult rat brain and suggest a function for neuregulins into adulthood.

Estradiol stimulates preoptic area-anterior hypothalamic proGnRH-GAP gene expression in ovariectomized rats

The decapeptide gonadotropin-releasing hormone (GnRH) and the 56-amino acid GnRH-associated peptide (GAP) are derived from a common precursor translated from the proGnRH-GAP mRNA. Studies using solid-phase hybridization techniques (i.e., Northern blot analysis, dot blot analysis, or in situ hybridization autoradiography) have yielded a controversy as to whether estradiol stimulates, inhibits, or has any effect on proGnRH-GAP gene expression in the preoptic area-anterior hypothalamus (POA-AH) of the ovariectomized (OVX) rat. Using a sensitive and quantitative solution hybridization-nuclease protection assay, which ensures complete hybridization of target RNA to probe RNA, we examined the effects of OVX and estradiol replacement on the amount of proGnRH-GAP mRNA in individual POA-AH dissections. Rats sacrificed at different intervals after OVX showed a significant time-dependent decrease (34-60%) in the levels of POA-AH proGnRH-GAP mRNA relative to sham-operated animals; OVX rats treated with estradiol, however, had proGnRH-GAP mRNA levels comparable to those of sham-OVX animals. To verify these observations, levels of the proGnRH-GAP peptide, measured by radioimmunoassay with antibodies directed against the cleavage and amidation site between the GnRH and the GAP portions fo the precursor molecular, were also found to decrease (37%) after OVX and increase (63-85%) following estradiol replacement, relative to intact rats. These data support the view that estradiol stimulates the levels of both proGnRH-GAP mRNA and its primary translation product in the POA-AH region of the OVX rat.

Acute intermittent nicotine treatment produces regional increases of basic fibroblast growth factor messenger RNA and protein in the tel- and diencephalon of the rat

Several findings show a neuroprotective effect of nicotine treatment in different experimental models, and a negative correlation has been observed between cigarette smoking and the incidence of Parkinsons disease. It seems possible that nicotine may in part exert its neuroprotective actions by favouring the synthesis of neurotrophic factors. The aim of this study was to determine whether the nicotine treatment could be associated with the induction of a neurotrophic factor in brain regions with nicotinic receptors. Thus, we analysed by in situ hybridization and RNAse protection assay the effects of (-)nicotine on basic fibroblast growth factor messenger RNA and by immunocytochemistry fibroblast growth factor-2 protein in the tel- and diencephalon of rats following single or acute intermittent (-)nicotine treatment. The present results showed that acute intermittent (-)nicotine treatment (four i.p. injections at intervals of 30 min), but not single injections, lead to a substantial and dose-related (0.1-2 mg/kg) up-regulation of fibroblast growth factor-2 messenger RNA levels in the cerebral cortex, in the hippocampus, in the striatum and ventral midbrain. This induction of fibroblast growth factor-2 expression peaked 4 h after the first injection and returned to normal levels within 24 h. The change of fibroblast growth factor-2 messenger RNA levels was associated with increased fibroblast growth factor-2 immunoreactivity mainly localized to nerve cells. The treatment was effective also when repeated in the same animals three or five days after the first injection. The pre-treatment with the non-competitive (-)nicotine receptor antagonist mecamylamine blocked the (-)nicotine effects on fibroblast growth factor-2 messenger RNA levels. In the above areas, no changes were observed in the fibroblast growth factor-1, 2 and 3 receptor messenger RNA levels nor in brain-derived neurotrophic factor messenger RNA levels. The present data indicate an ability of intermittent (-)nicotine to increase fibroblast growth factor-2 in many tel- and diencephalic areas. In view of the trophic function of fibroblast growth factor-2, the previously observed neuroprotective effects of (-)nicotine may at least in part involve an activation of the neuronal fibroblast growth factor-2 signalling, and open up new avenues for treatment of Parkinsons disease and Alzheimers disease based on the existence of nicotinic receptor subtypes enhancing fibroblast growth factor-2 signalling in many regions of the tel- and diencephalon.

Glutamate regulation of GDNF gene expression in the striatum and primary striatal astrocytes

The aim of this study was to investigate the regulation of glial cell line-derived neurotrophic factor (GDNF) mRNA by activation of glutamate receptors in the rat striatum. We observed an increase in GDNF mRNA levels in the adult rat striatum after administration of subseizure doses of N-methyl-D,L-aspartic acid (NMA) and kainic acid. Since it is unclear whether the upregulation of GDNF occurred in neurons or astrocytes within the striatum, we further investigated whether GDNF gene expression in primary striatal astrocytes in culture could be regulated by glutamate receptor activation. We found that treatment of the cultures with NMA and kainic acid similarly upregulated GDNF gene expression as observed in vivo, suggesting that striatal astrocytes express functional glutamate receptors. Immunocytochemical and nuclease protection analysis revealed that striatal astrocytes expressed the NMDA-R1 subunit. These findings suggest the regulation of GDNF mRNA in the striatum may be mediated by excitation of glutamate receptors via glutamatergic cortical afferents.

Prenatal corticosterone increases spontaneous and d-amphetamine induced locomotor activity and brain dopamine metabolism in prepubertal male and female rats

Recently, both glucocorticoid receptor immunoreactivity and glucocorticoid receptor messenger RNA levels were found in multiple brain areas, especially in the neuroepithelium during the late prenatal development of the rat brain. To better understand the potential influence of stress on fetal brain development by release of maternal adrenocortical steroids, we have investigated the effects of corticosterone administration to pregnant rats on the locomotor activity of their prepubertal offspring. On day 16 of pregnancy female rats were implanted with either placebo or corticosterone pellets (release of 2.4 mg/day for seven days). After birth their offspring were nursed by foster mothers to avoid any postnatal effects of the corticosterone pellets. At three weeks of age, the offspring were tested for spontaneous motor behaviours. Both male and female offspring from corticosterone treated mothers showed significantly increased spontaneous ambulation, motility and rearing compared to placebo treated groups. No significant sex differences were found in locomotor activity between male and female offspring from placebo groups. Following d-amphetamine (1.5 mg/kg) treatment, a preferential dopamine releasing agent, we observed a significant increase in ambulation, motility and rearing activity in the male offspring treated with corticosterone. In the female offspring, only the rearing activity was significantly higher after d-amphetamine treatment in the prenatal corticosterone group compared with the placebo treated group. Basal dopamine metabolism (dihydroxyphenylacetic acid/dopamine ratio) was increased in the dorsal striatum and ventral striatum of male and female offspring from corticosterone-treated dams. In the male offspring, corticosterone treatment was associated with a disappearance of the right side dominance of dopamine metabolism in the dorsal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor and Basic Fibroblast Growth Factor Protect Dopaminergic Neurons from Glutamate Toxicity in Culture

Abstract: In this report we characterize the toxicity of the excitatory amino acid l‐glutamate with respect to dopaminergic neurons cultured from embryonic rat mesencephalon. We also demonstrate that two growth factors, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), can protect these neurons from damage. Micromolar concentrations of l‐glutamate, as well as agonists that specifically activate N‐methyl‐d‐aspartate (NMDA) and non‐NMDA receptors, are all toxic to dopamine neurons in a concentration‐dependent manner, as reflected by decreases in high‐affinity dopamine uptake and confirmed by decreases in numbers of tyrosine hydroxylase‐immunoreactive neurons. Although the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione could attenuate the effects of quisqualate, treatment with this antagonist could not eliminate the effects of glutamate itself. Similarly, (±)‐2‐amino‐5‐phosphonopentanoic acid was effective against NMDA toxicity but could not protect cells from quisqualate toxicity. Thus, each type of receptor could mediate neurotoxicity independently of the other. The presence of EGF or bFGF in the culture medium conferred a relative resistance of dopaminergic neurons to glutamate and quisqualate neurotoxicity by increased glutamate transport. However, treatment of the cultures with l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid, an inhibitor of glutamate transport, attenuated but did not eliminate the protective effects of both growth factors against glutamate toxicity. When cultures were incubated with conditioned medium from growth factor‐treated cultures, neuroprotection was also achieved. These results suggest that both EGF and bFGF can protect neurons from neurotoxicity in culture by increasing the capacity of the culture for glutamate uptake as well as by the secretion of soluble factors into the medium.