Françoise Robert

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

Without medical progress, dementing diseases such as Alzheimers disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimers disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimers patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimers disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimers patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Steroid synthesis and metabolism in the nervous system: Trophic and protective effects

Steroids influence the activity and plasticity of neurons and glial cells during early development, and they continue to exert trophic and protective effects in the adult nervous system. Steroids are produced by the gonads and adrenal glands and reach the brain, the spinal cord and the peripheral nerves via the bloodstream. However, some of them, named “neurosteroids”, can also be synthesized within the nervous system. They include pregnenolone, progesterone, dehydroepiandrosterone and their reduced metabolites and sulfate esters. Little is known concerning the regulation of steroid synthesis in the nervous system, which involves interactions between different cell types. For example, the synthesis of progesterone by Schwann cells in peripheral nerves is regulated by a diffusible neuronal signal. Neurotrophic and neuroprotective effects of steroids have been documented both in cell culture and in vivo. PROG plays an important role in the neurological recovery from traumatic injury of the brain and spinal cord by mechanisms involving protection from excitotoxic cell death, lipid peroxydation and the induction of specific enzymes. After transection of the rat spinal cord, PROG increases the number of nitric oxide synthase expressing astrocytes immediately above and below the lesion. PROG also plays an important role in the formation of new myelin sheaths. This has been shown in the regenerating mouse sciatic nerve after lesion and in cocultures of sensory neurons and Schwann cells. PROG promotes myelination by activating the expression of genes coding for myelin proteins. The modulation of neurostransmitter receptors, in particular the type A γ-aminobutyric acid, the N-methyl-D-aspartate and the sigma 1 receptors, is involved in the psychopharmacological effects of steroids and allows to explain their anticonvulsant, anxiolytic, antidepressive and sedative effects as well as their influence on memory. Pregnenolone sulfate has been shown to reverse age-related deficits in spatial memory performance and to have protective effects on memory in different models of amnesia.

Progesterone synthesis and myelin formation in peripheral nerves

Progesterone is synthesized in the nervous system by neurons and glial cells. Because of their simple structure, plasticity and capacity of regeneration, peripheral nerves are particularly well suited for studying the biosynthesis, mechanisms of action and effects of the hormone. Schwann cells, the myelinating glial cells in the peripheral nervous system, synthesize progesterone in response to a diffusible neuronal signal. In peripheral nerves, the local synthesis of progesterone plays an important role in the formation of myelin sheaths. This has been shown in vivo, after cryolesion of the mouse sciatic nerve, and in vitro, in cocultures of Schwann cells and sensory neurons. Schwann cells also express an intracellular receptor for progesterone, which thus functions as an autocrine signalling molecule. Progesterone may promote myelination by activating the expression of genes coding for transcription factors (Krox-20) and/or for myelin proteins (P0, PMP22). Recently, it has been proposed that progesterone may indirectly regulate myelin formation by influencing gene expression in neurons. Steroid hormones also influence the proliferation of Schwann cells: estradiol becomes a potent mitogen for Schwann cells when levels of cAMP are elevated and glucocorticosteroids have been shown to increase the mitogenic effects of peptide growth factors.

Neurosteroids: Expression of Functional 3β-Hydroxysteroid Dehydrogenase by Rat Sensory Neurons and Schwann Cells

Steroids which are synthesized within the nervous system, such as progesterone, have been termed ‘neurosteroids’. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3β‐hydroxysteroid dehydrogenase and can convert [3H]pregnenolone to [3H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [3H]pregnenolone, they produce 5–10 times more [3H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5α‐dihydroprogesterone, but unlike Schwann cells, they do not produce 3α,5α‐tetrahydroprogesterone, a potent positive allosteric modulator of γ‐aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [3H]pregnenolone to progesterone and its 5α‐reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.

The Neurohormonal Thymic Microenvironment: Immunocytochemical Evidence that Thymic Nurse Cells Are Neuroendocrine Cells

Thymic neuroendocrine cells were identified by immunofluorescence in the murine thymus through the use of monoclonal antibody A2B5, and specific polyclonal antisera against neurophysin (NP), oxytocin (OT) and arginine vasopressin (AVP). Two reactive regions were clearly identified: the subcapsular cortex and the medulla. A close correspondence was observed between A2B5-reactive and NP-immunoreactive cells in the medulla. An important epithelial population of the subcapsular cortex, the thymic nurse cells (TNCs), were found to be A2B5-positive and to contain immunoreactive NP, OT and AVP. The neuroendocrine nature of TNCs was further substantiated by their high reactivity with an antiserum against neuron-specific enolase. These observations demonstrate the presence in the thymus gland of an original neuroendocrine microenvironment which could be of functional importance in the mediation of central influences upon T lymphocyte differentiation.

Genomic and membrane actions of progesterone: implications for reproductive physiology and behavior

Progesterone, produced by the ovaries and adrenal glands, regulates reproductive behavior and the surge of luteinizing hormone which precedes ovulation by acting on neurons located in different parts of the hypothalamus. The study of the activation of these reproductive functions in female rats has allowed to explore the different mechanisms of progesterone action in the brain. It has allowed to demonstrate that new actions of the hormone, which have been observed in particular in vitro systems, are also operational in vivo, and may thus be biologically relevant. This mainly concerns the direct actions of progesterone on receptors of neurotransmitters such as oxytocin and GABA. Activation of the progesterone receptor in the absence of ligand by phosphorylation may also play a role.

Synthesis of progesterone in Schwann cells: regulation by sensory neurons

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3β‐hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3β‐hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3β‐hydroxysteroid dehydrogenase were induced. Regulation of 3β‐hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3β‐hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re‐expressed. After nerve transection, which does not allow axonal regeneration, 3β‐hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3β‐hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.

COLOCALIZATION OF IMMUNOREACTIVE OXYTOCIN, VASOPRESSIN AND INTERLEUKIN-1 IN HUMAN THYMIC EPITHELIAL NEUROENDOCRINE CELLS

Monoclonal antibodies to oxytocin (OT) and vasopressin (VP) revealed some positively staining stromal cells in the subcapsular cortex and in the medulla of the human thymus. We further demonstrated that these cells are a subset of epithelial endocrine cells and also contain immunoreactive interleukin-1 together with the neuropeptides. In addition, the thymic cells stained by monoclonal antibodies directed to the cyclic part of oxytocin or vasopressin also contained some immunoreactive neurophysins. These data support the concept of intrathymic synthesis of neurohypophyseal-like peptides fitting the hypothalamic model. However, we observed that, contrary to the situation in the brain, OT- and VP-like peptides colocalized in the same thymic cells. Furthermore, one monoclonal antibody, specific for the tail part of oxytocin, did not label thymic cells. Therefore, thymic neuropeptide(s) could be related to, but distinct from, authentic OT and VP. These observations suggest some molecular differences between hypothalamic and thymic oxytocin biosynthetic pathways which need to be further investigated.

The neuroendocrine thymus

SummaryCertain subtypes of thymic epithelial cells — the medullary epithelium, the cortical surface epithelium, and some intracortical epithelial cells — show strong immunohistochemical reactivity with antisera against oxytocin, Argvasopressin and neurophysin. The epithelial nature of the neuropeptide containing cells is shown by their morphology and their reactivity with monoclonal anti-cytokeratin AE1/E3. Hassalls corpuscles are positive as well. The immunoreactivity patterns for the three neuropeptides are identical, suggesting a parallel distribution. The vast majority of cortical epithelial cells are negative, emphasizing the tightly controlled microenvironment for T-cell development. The possibility of a neuroendocrine role of the thymus is discussed.

Comparison of three immunoassays in the screening and characterization of monoclonal antibodies against arginine-vasopressin *

A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.