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The role of neurosteroids in healthy ageing: therapeutical perspectives

Deliverables

Relations between neurosteroid levels within distinct brain regions and cognitive performance, attention and motivation in old rats have been established. This has required the development of a new, very sensitive behavioural test for attention and motivation. These experiments will be pursued to determine whether changes in steroid levels during early life are predictive for later cognitive impairments. Neurosteroids influence cognition, attention and motivation by modulating the activity of cholinergic neurons in the fore- and hinbrain. Cholinergic neurons of the medial septum, which project to the hippocampus, are involved in the promnesic effects of pregnenolone sulfate. When infused into the medial septum, pregnenolone sulfate increased spatial memory performance and enhanced acetylcholine release within the dorsal hippocampus. Neurosteroids also modulate the activity of cholinergic neurons within the peduncolopontine tegmental nucleus (PTT) of the posterior brain. Morphological alterations of the cholinergic neurons of the PTT have been related to the memory performance of old rats. These studies have also provided evidence for the first time that mild cognitive deficits observed in old subjects are clearly associated with alterations of the circadian rhytmicity, mainly reflecting fragmentation of sleep parameters.
A new unction for GABAA receptors has been discovered, with likely consequences for the prevention and treatment of age-related myelin abnormalities. Schwann cells express GABAA receptors and the modulation of their activity plays an important role expression of the peripheral myelin protein PMP22. The stimulatory effects of 3a,5a-tetrahydroprogesterone and of 3a,5a-tetrahydrotestosterone (3adiol) on the PMP22 gene can indeed be mimicked by the selective GABAA receptor agonist muscimol and blocked by the selective antagonist bicuculline. How Schwann cell GABAA receptors regulate myelination still needs to be elucidated. 3a,5a-tetrahydroprogesterone also promotes myelination via the modulation of GABAA receptors within the CNS, as shown in cerebellar explant cultures of P7-rats. However, although the modulation of GABAA receptors by 3a,5a-tetrahydroprogesterone obviously plays a role in myelination, binding of PROG to its intracellular receptor appears to be required, as the promyelinating effects of progestins were completely blocked by the progesterone receptor antagonist RU486 and as progesterone had no effect on myelin formation in cerebellar slices from PRKO mice. One mechanism by which the activation of GABAA receptors by 3a,5a-tetrahydroprogesterone may accelerate myelination in the brain is the stimulation of oligodendrocyte progenitor proliferation. This neurosteroid increased the proliferation of ePSA-NCAM+ neural progenitors grown in "oligospheres" via membrane GABAA receptors.
Neural precursors located in the subgranular zone of the dentate gyrus of the hippocampus proliferate in adult rodents. The newly generated neurons are functional, integrate in hippocampal circuits and may be involved in certain forms of hippocampal-dependent learning. In the hippocampus of aged animals, the administration of neurosteroids can still promote neurogenesis in a long-lasting manner. In addition, a clear relationship was observed between the cognitive performances of old rats and the formation of new neurons in the hippocampus. The GABA active neurosteroids pregnenolone sulphate and 3?,5?-tetrahydroprogesterone respectively stimulated and inhibited the proliferation of neuronal progenitors. Their effects indeed involved GABAA receptors, as demonstrated in cultured "neurospheres" by using selective GABAA receptor agonists and antagonists. Estradiol also modulates, by interacting with the growth factor IGF-1, adult neurogenesis in the hippocampus. The actions of estradiol and IGF-1 were shown to converge on the intracellular AKT/PKB signalling pathway.
The influence of GABAA receptor subunit composition on the GABA-modulatory effects of pregnane steroids has been investigated by partner 4 using the Xenopus laevis oocyte expression system and the two electrode voltage clamp technique. Studies of the interactions of 3a, 5a-tetrahydroprogesterone with 14 different GABAA receptor isoforms have been completed show the subunit composition to be an important determinant of neurosteroid action. The subunit composition of GABAA receptors may thus contribute to their neuronal specificity and behavioural profile. An important finding was that not only the subunit composition, but also phosporylation by protein kinase C (PKC) could influence the interaction of pregnane steroids with synaptic GABAA receptors. The interaction of neurosteroids with GABAA receptors is brain region and indeed neurone dependent and the activity of neuronal kinases contribute to this heterogeneity, and potentially provide a mechanism to dynamically influence the actions of neurosteroids. Experimental evidence showed that the PKCe isoform is involved in governing the neurosteroid/ GABAA receptor interactions. The molecular interactions of the PKCe with GABAA receptor subunits is currently under investigation.
Histological analysis revealed that progesterone has no deleterious effects on the rapid spontaneous remyelination in young male rats. Treatment with progesterone had subtle beneficial effects on the slow and inefficient remyelination in the brains of old male rats. Progestins may promote remyelination by modulating inflammatory responses or by stimulating the proliferation of progenitors cells. An important finding was the role of gender in CNS remyelination. Thus, in the brain, the rate of remyelination declines more rapidly with aging in males than in females.
Transgenic mice have been generated to study the regulation of PMP22. This peripheral myelin protein is the culprit in the vast majority of motor and sensory neuropathies, which show an increased frequency in the elderly. In addition, some of the changes which peripheral nerves undergo during normal ageing are similar to those observed in peripheral neuropathies. The 10 kilobases of 5'-flanking region of the PMP22 gene contains the necessary information to mirror its endogenous expression pattern in peripheral nerves during development and regeneration and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination, and remyelination in Schwann cells, demonstrating the crucial influence of neuron-Schwann cell interactions in the regulation of PMP22. During the last reporting period, regulatory regions of the PMP22 promoter that direct the temporal, spatial and cell-specific expression of the myelin protein have been identified. The mice also offer an unique model to investigate the regulation by neurosteroids of PMP22 expression, not only in cultured cells, but also in vivo.
To better understand the pathophysiological significance of neurosteroids, it was necessary to develop a new method for the accurate measure of their low levels within small samples of nervous tissue. With the support of the European Community (Biomed 2 contract BMH4972359), partner 1 has developed a very sensitive micro-assay, allowing to accurately measuring low steroid levels in small tissue samples by gas chromatography/mass spectrometry (GC/MS) (http://cordis.europa.eu/marketplace). His laboratory thus became a "Centralized European Facility" for the measure of neurosteroids. Within the frame of the present project, reference values for steroid levels in rat, mouse and human nervous tissues and plasma have been provided by gas chromatography/mass spectrometry (GC/MS). The concentrations of steroids measured by GC/MS technology not always correspond to the ones previously determined by less accurate assay procedures such as radioimmunoassays. The "brain cartography" of neurosteroids has been established for the rat and mouse and the neuroanatomical distribution and developmental changes in the expression of the 3b-hydroxysteroid dehydrogenase, enzyme, which converts pregnenolone to progesterone, has been described. Neurosteroid levels were found to change with age and in response to injury. Large amounts of lipoidic derivatives of pregnenolone and dehydroepiandrosterone have been identified in rat and human brain tissue and plasma. This finding may have important consequences for our understanding of neurosteroid functions. Neurosteroid levels and metabolism have also been studied in the aged human brain (non-demented and Alzheimer's patients). A general trend toward decreased levels of all steroids was observed in all Alzheimer's patients¡¦ brain regions compared with controls. High levels of key proteins implicated in the formation of plaques and neurofibrillary tangles (fO-amyloid peptides, phosphorylated tau) were correlated with decreased brain levels of PREGS and DHEAS, suggesting a possible neuroprotective role of these neurosteroids in AD.
Two complementary therapeutic strategies to promote successful aging have been explored within the frame of this project: - The systemic administration of steroids and - The stimulation of the local synthesis of neurosteroids in the nervous system by ligands of the mitochondrial benzodiazepine receptor (MBR). Indeed, steroids present in nervous tissues originate either from the steroidogenic endocrine glands (gonads and adrenal glands) as they easily cross the blood-brain barrier, or from local synthesis. It is in fact now well established that some steroids, which have been named "neurosteroids", can be synthesized in the brain, spinal cord and peripheral nerves by neurons and glial cells. A rate-limiting step in steroid synthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cytochrome P450scc, enzyme that converts cholesterol to pregnenolone, is located. The MBR is a protein of the outer mitochondrial membrane, necessary for the intramitochondrial transport of cholesterol. The administration of MBR agonists has been shown to increase the synthesis of steroid hormones by the gonads and of neurosteroids within the nervous system. It is important to note that the MBR, also called "peripheral benzodiazepine receptor" (PBR), is distinct from the classical benzodiazepine sites located on GABAA receptors: the MBR it is not associated with GABAA receptors and recognizes selective ligands. The administration of the MBR agonist allows to protect neurons and to reverse age-related abnormalities of myelin sheaths. Thus, hilar neurons of the hippocampus can be protected against excitotoxic injury by the administration of Ro5-4864. Activation of the MBR receptor has also beneficial effects on ageing-associated degeneration of the rat sciatic nerve, increasing the total number of myelinated fibers and decreasing the percentage of fibers with myelin decompaction. All these effects were blocked by the simultaneous treatment with the MBR antagonist PK-11195. The effects of the MBR ligands apparently did not involve changes in neurosteroid levels and are distinct from those previously observed in response to the systemic administration of progestins. Attempts to stimulate the synthesis of neurosteroids by MBR ligands in nervous tissues of old rats have not been successful so far, suggesting an impairment of MBR-induced steroidogenesis in old animals. There is an obvious need for more efficient MBR ligands. Two novel MBR ligands have been studied in collaboration with two industrial partners.
Neurosteroids and MBR ligands were shown to have neuroprotective effects after excitotoxic brain injury, to which aged brains are particularly vulnerable. Treatment with 3a, 5a-tetrahydroprogesterone not only affected the survival of hippocampal neurons, but also influenced the synaptic remodelling after neuronal loss. The effect seems to be specific for certain type of synapses, as it does differently affect the GABAergic and non-GABAergic terminals. A significant loss of hilar neurons in the hippocampus of male and female rats has been observed between 22 and 24 months of age. The administration of progestins resulted in a significant reduction in neuronal loss at this age. By using unbiased stereological quantification methods, it was shown that the ageing process of the hippocampus is not only accompanied by neuronal death, but also by changes in synaptic connectivity. Estradiol also rescued hippocampal neurons after excitotoxic injury, and regulated adult neurogenesis in the hippocampus by acting in synergy with the growth factor IGF-1. The actions of the estradiol and IGF-1 were shown to converge on the intracellular AKT/PKB signalling pathway. The local formation of estradiol by the aromatase enzyme may play an important role.
Age-related changes in peripheral nerves mainly affect the myelinated fibers. Messenger and protein levels of the major peripheral myelin proteins P0 and PMP22 were found to be decreased during ageing. In the sciatic nerves of old rats, treatment with 5a-dihydroprogesterone increased P0 mRNA levels, both progesterone and 5a-dihydroprogesterone increased P0 protein and the administration of 3a, 5a-tetrahydroprogesterone increased PMP22 protein. In contrast to progestins, androgens did not affect neither P0 nor PMP22 expression in the sciatic nerve of aged rats. Progesterone is not only involved in the control of myelination in peripheral nerves, but also in axon maintenance. The prolonged administration of progestins allowed the reversal of age-related structural abnormalities of the peripheral myelin sheaths. The systemic treatment of old male rats with progesterone, 5a-dihydroprogesterone or 3a,5a-tetrahydroprogesterone significantly decreased the percentage of fibers with myelin abnormalities and the number of fibers with irregular shapes and it increased the number of small myelinated fibers. Consistent with the finding that androgens did not affect P0 or PMP22 expression, their administration was also inefficient in reversing the myelin abnormalities. This is the first demonstration that age-relate abnormalities of myelin sheaths can be reversed by chronic treatment with neurosteroids. Activation of the mitochondrial benzodiazepine receptor (MBR) is also able to exert beneficial effects on ageing-associated degeneration of the rat sciatic nerve. These findings thus support the potential role of MBR agonists to prevent aging-associated peripheral nerve degeneration.
A novel concept, which has emerged, is the existence of a tonic neurosteroid tone within brain regions such as hippocampus, which is critically involved in cognitive processes. Thus, interactions of endogenous 3a, 5a-tetrahydroprogesterone with GABAA receptors was promoted by application of two types of inhibitors: - Of the SULT1A1, a sulfotransferase which converts biologically active 3a, 5a-tetrahydroprogesterone to its biologically inactive sulfated form ; - Of the 3a-HSOR, an enzyme involved in the synthesis and degradation of 3a, 5a-tetrahydroprogesterone. Application of these inhibitors indeed resulted in prolonged miniature inhibitory postsynaptic currents (mIPSCs) recorded from hippocampal neurons. By contrast, these inhibitors did not directly interact with the GABAA receptors.

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