Steroids and neuroregeneration

Analysis and profiling of steroids by gas chromatography/tandem mass spectrometry

PI : Philippe Liere (Team 1), Michael Schumacher (Dir Team 1)

We analyse steroid metabolomes in plasma and small nervous tissue samples by gas chromatography-tandem mass spectrometry (GC-MS/MS). This technology represents the gold standard for accurate, sensitive and extensive steroid profiling.The GC-MS/MS analysis of steroids is an evolutive technology, and our group is engaged in further development. So far, GC-MS/MS allows us the profiling of up to 70 different steroids, in the femtomole range, in a robust and precise manner in small tissue and biological fluids samples.

However, this technology only provides accurate reference values when combined with upstream very careful samples purification and fractionation methods in order to obtain a reliable steroid profiling without cross-contamination The combination between validated sample preparation and a reference analytical technology is a prerequisite for steroid analysis in biological samples, and in particular in complex matrices such as fatty nervous tissues. Radioimmunoassays and enzyme-linked immunosorbent assays, even with prepurification steps, do not provide the required specificity. The major strength of GC-MS/MS is its capacity to separate a large number of structurally similar steroids such as stereoisomers and enantiomers, thus permitting to obtain key informations on the neuroendocrine physiopathological processes in injuried nervous system, in neurodegenerative diseases.

We provide steroid analysis for academic, preclinical and clinical research projects as well as for pharmaceutical companies.

Role of neurosteroid synthesis in cerebroprotection after ischemic stroke

PI : Rachida Guennoun (Team 1)

The project aims to better understand the role of neurosteroid synthesis in cerebroprotection after ischemic stroke and the mechanisms by which steroids exert their cerebroprotective effects at the molecular and cellular level.

We have a multidisplinary approach combining generation of mice with selective invalidation of steroid receptors or enzymes of steroidogenesis using the Cre/Lox strategy, surgery (experimental cerebral ischemia), behavioral tests to evaluate functional recovery (neurological deficits, motor coordination), histological analysis, gene and protein expression analysis (Transcriptomic analysis, RT-qPCR, Western blot, immunofluorescence, Confocal microscopy) and steroid measurement by Gas Chromatography-tandem mass spectrometry.

Thanks to the interaction between basic scientists and clinicians we aim to generate preclinical data for the development of a neuroprotective therapy based on treatment with natural or synthetic steroids or on stimulation of endogenous synthesis of neurosteroids in stroke patients of both sexes.

New steroid-associated therapeutic approaches for myelin diseases in the central nervous system

PIs : Elisabeth Traiffort (Team 1), Abdel Ghoumari (Team 1)

In the central nervous system, oligodendrocytes form insulating myelin sheaths around axons, ensuring rapid electrical communication between neurons and providing trophic support. Damaged myelin can be partly replaced by spared oligodendrocytes and the recruitment of oligodendrocyte progenitor cells (OPC), followed by their differentiation into myelinating oligodendrocytes. This process is known as remyelination, myelin regeneration or myelin repair. In demyelinating diseases such as multiple sclerosis, remyelination often remains insufficient or fails, and enhancing the regenerative capacity of myelin is now regarded as a promising therapeutic option.

When considering remyelination therapies, an important but often neglected factor is the hormonal environment of neural cells. Steroid hormones are indeed important regulators of the proliferation and differentiation of OPC and myelin formation by interacting with pro-myelination signaling pathways such as Hedgehog signaling. Moreover, reduced levels of gonadal steroids in multiple sclerosis patients have been associated with neurological disability and worse clinical outcomes.

Cooperation of the androgen and hedgehog signaling pathways during myelination.

PI : Elisabeth Traiffort (Team 1)

Our current projects aim to dissect the molecular and cellular mechanisms involved in the processes of myelin production by oligodendrocytes during development and following demyelination of the central nervous system. Two signaling pathways are currently under investigation. These are the pathways induced respectively by the steroid hormone testosterone and by secreted Hedgehog proteins. The project is based on the genetic and pharmacological manipulation of these pathways during development and in different mouse models of demyelination.

The group of E. Traiffort recently demonstrated a cooperation between testosterone and Hedgehog signaling in myelination. Both pathways exert complementary activities during postnatal oligodendrogenesis. In adults, their cooperation promotes the differentiation of OPC into mature myelin forming oligodendrocytes, as well as a higher preservation of axonal integrity and lower neuroinflammation. In experimental autoimmune encephalomyelitis (EAE), an animal model most commonly used to investigate multiple sclerosis pathology, both pathways ameliorate disease symptoms in a concerted manner.

Laouarem Y, Kassoussi A, Zahaf A, Hutteau-Hamel T, Mellouk A, Bobé P, Mattern C, Schumacher M, Traiffort E (2021) Functional cooperation of the hedgehog and androgen signaling pathways during developmental and repairing myelination. Glia 69:1369-1392.

The role of steroid hormones in myelination and the sexual differentiation of myelin.

Our laboratory has been pioneering in demonstrating a role of steroid hormones in myelination. Pro-myelinating actions of progesterone have been first demonstrated for Schwann cells in the peripheral nervous system, and then for oligodendrocytes in the central nervous system. These were among the first observations of steroid actions in the nervous system going beyond classical reproductive functions. The impact of the steroid hormonal milieu on the myelination process may have its roots in the concomitant evolutionary appearance of myelin and the diversification of steroid receptors in hinge-jawed vertebrates.

Testosterone can exert persistent differentiating effects on the brain, qualified as organizational, by acting on immature neuronal circuits during sensitive developmental periods.

PI : Abdel Ghoumari (Team 1)

We have shown that neonatal testosterone, acting via brain androgen receptors (AR), exerts persistent organizing effects on the structure of myelin sheaths in the male mouse brain. As a result, adult male mice have thicker myelin sheaths, a greater density of oligodendrocytes and shorter myelinated axonal segments (internodes) when compared with females.

Structural sex differences in the myelin sheaths may impact on their integrity and vulnerability to immune attacks and may contribute sex-related differences in the incidence and progressions of demyelinating diseases such as multiple sclerosis.

Abi Ghanem C, Degerny C, Hussain R, Liere P, Pianos A, Tourpin S, Habert R, Macklin WB, Schumacher M, Ghoumari AM (2017) Long-lasting masculinizing effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS Genet 13:e1007049.

PI : Elisabeth Traiffort (Team 1)

It is well-known that Testosterone is a male hormone produced by the testes. However, it is less appreciated that testosterone, at lower levels, is also en essential hormone in women. So far, the remyelinating, neuroprotective and anti-inflammatory actions of testosterone have been studied in male mice and men suffering from multiple sclerosis. However, androgen effects mediated by the androgen receptor (AR) have been only poorly studied in females. Recently, we have shown significant AR expression in demyelinated lesions from female mice and women with multiple sclerosis. In female mice, androgens and estrogens act in a synergistic way while androgens drive microglia responses towards myelin regeneration. Our work also uncovered major sex-dependent molecular differences regarding androgen effects during remyelination. Most genes down-regulated by androgens in female mice are related to inflammation, whereas those predominantly down-regulated in males are related to lipid metabolic processes. Thus, androgens are essential for proper myelin regeneration in females and therapeutic approaches of demyelinating diseases need to consider male-female differences.

Zahaf A, Kassoussi A, Hutteau-Hamel T, Mellouk A, Marie C, Zoupi L, Tsouki F, Mattern C, Bobé P, Schumacher M, Williams A, Parras C, Traiffort E (2023) Androgens show sex-dependent differences in myelination in immune and non-immune murine models of CNS demyelination. Nature Communications 14:1592.

Protection from tau pathology through TSPO-related regulation of steroid synthesis

PI: Yvette Akwa (Team 1), Davide Tampellini (Baulieu Institute, Team 1), Michael Schumacher (Team 1)

The progressive hyperphosphorylation and aggregation of tau proteins in the brain forms aberrant filamentous inclusions giving rise to neurofibrillary tangles (NFT), the feature of tauopathies. The presence of tau aggregates is associated with synaptic loss, mitochondrial impairments and neuroinflammation.  The 18 kDa translocator protein (TSPO/PBR) is a mitochondrial membrane protein that participates in steroidogenesis. Many studies including ours showed that TSPO ligands stimulate the endogenous production of neuroactive steroids in mice and rats.  Additionally, besides its implication in neuroinflammation, published data indicate increased neuronal TSPO expression upon stimulation of neuronal activity. We hypothesized that TSPO ligands XBD173 and etifoxine treatment ameliorate tau hyperphosphorylation, tau oligomer clearance and impaired mitochondrial structure/activity in P301L cells and P301S mouse models of FTDP-17, by involving the stimulation of neurosteroidogenesis and/or TFEB activation. Complete deletion of tspo/pbr gene in P301L cells will show specific effects of TSPO ligands. Over the past decades, the use of genetic TSPO-knockout models has challenged the function of TSPO/PBR in steroid biosynthesis. We recently show that TSPO is not required for the novo synthesis of pregnenolone in the mouse adrenal glands and brain under basal conditions. A steroidome in wild-type TSPO-KO mice and TSPO-KO rats treated with etifoxine is under investigation. Overall, the modulation of TSPO/PBR activity could represent an attractive disease-modifying strategy in tauopathies.

Further studies are ongoing to evaluate the effects of direct administration of natural steroids or their synthetic enantiomers, particularly excitatory compounds stimulating neurotransmission.

El Chemali L, Akwa Y, Massaad-Massade L. The mitochondrial translocator protein (TSPO): a key multifunctional molecule in the nervous system. Biochem J. 2022, 479:1455-1466

Regulation of inhibitory synapses by neuroactive steroids (NAS): structure-function relationships

PI: Christian Specht (Team 1)

Neuroactive steroids (NAS) are promising pharmacological agents for the treatment of depression and stress-related pathologies. Their sedative and anticonvulsant effects result primarily from allosteric modulation of inhibitory GABA-A receptors, causing increased inhibitory activity. However, the mechanisms by which NAS regulate network activity are not known due to the multitude of molecular targets as well as the lack of information on the short- and long-term effects of NAS on the distribution of receptors in the neuronal membrane and the concomitant adaptive changes in inhibitory neurotransmission. We systematically analyze the effects of GABA-AR-specific NAS with known clinical profiles on inhibitory neurotransmission in cultured primary neurons. Neurons are exposed to NAS for different durations to determine the short- and long-term effects of NAS at inhibitory synapses and in the initial segment of the axon (AIS), focusing on the balance between receptors synaptic and extrasynaptic. We will characterize the effects of NAS treatment on the subcellular distribution of GABA-ARs and glycine receptors (GlyRs) using conventional and super-resolution fluorescence imaging, as well as the consequences on inhibitory neurotransmission using electrophysiological measurements under control conditions and in response to patient-derived autoantibodies against inhibitory receptor subunits. Our project contributes to bridging the gap between the short-term regulation of GABAergic and glycinergic currents by NAS and the long-term adaptation of the network by homeostatic or anti-homeostatic processes.