Ayikoe Guy Mensah-Nyagan

Evidence for a key role of steroids in the modulation of pain

Neurotransmitters such as glutamate, substance P, serotonin and gamma-aminobutyric acid pivotally control pain mechanisms. It is also well known that inflammatory and/or neuropathic pain may depend on the action of diverse cytokines and other molecules including eicosanoids, endorphins, calcitonin-gene related peptide, free radicals and transcription factors. Because steroids control the development, activities and plasticity of the nervous system, these compounds are of particular interest in the modulation of pain. The paper discusses various data supporting the existence of key regulatory effects of steroids in the control of pain. In particular, we analyzed three categories of observations which historically contributed to demonstrate that endogenous and synthetic steroids play a crucial role in the regulation of neurobiological processes involved in pain sensation. The first series of data, which present the chemical characteristics enabling steroids to act on several tissues, also summarize pertinent results supporting the modulation of pain sensation by steroidal compounds. The second category of data evokes psychosocial, fundamental and clinical results suggesting the existence of sex steroid-based differences in pain perception. Finally, we discuss recent evidence showing the endogenous production of neurosteroids and their effects in the spinal cord which crucially controls pain transmission. Taken together, the data reviewed herein suggest that future investigations aiming to develop effective steroid-based strategies against chronic pain must integrate in a complementary manner anti-inflammatory properties of steroids, sex steroid-induced dimorphism in pain perception and regulatory effects exerted by endogenous neurosteroids in pain neural circuits.

Neurogenic pain and steroid synthesis in the spinal cord.

The spinal cord (SC) is a biosynthetic center for neurosteroids, including pregnenolone (PREG), progesterone (PROG), and 3α/5α-tetrahydroprogesterone (3α/5α-THP). In particular, anactive form of cytochrome P450 sidechain cleavage (P450scc) has been localized in sensory networks of the rat SC dorsal horn (DH). P450scc is the key enzyme catalyzing the conversion of cholesterol (CHOL) into PREG, the rate-limiting step in the biosynthesis of all classes of steroids. To determine whether neurosteroidogenesis might be involved in the pivotal role played by the DH in nociception, effects of neurogenic pain provoked by sciatic nerve ligature were investigated on P450scc expression, cellular distribution, and activity in the SC. P450scc mRNA concentration was threefold higher in the DH of neuropathic rats than in controls. The nerve ligature also increased the density of P450scc-positive neuronal perykarya and fibers in the ipsilateral DH. Incubation of spinal tissue homogenates with [3H]CHOL revealed that the amount of newly synthesized [3H]PREG from [3H]CHOL was 80% higher in the DH of neuropathic rats. Radioimmunoassays showed an increase of PREG and 3α/5α-THP concentrations in neuropathic rat DH. The upregulation of PREG and 3α/5α-THP biosynthesis might be involved in endogenous mechanisms triggered by neuropathic rats to cope with the chronic pain state. 3α/5α-THP formation from PREG can also generate PROG, which decreases sensitivity to pain and protects nerve cells against degeneration. Because apoptotic cell death has been demonstrated in the DH during neuropathic pain, activation of neurosteroidogenesis in spinal tissues might also be correlated to the neuroprotective role of steroids in the SC.

Allopregnanolone prevents and suppresses oxaliplatin-evoked painful neuropathy: Multi-parametric assessment and direct evidence

&NA; Oxaliplatin (OXAL) is a platinum‐based drug used for the treatment of colorectal, lung, breast and ovarian cancers. OXAL does not cause renal or hematologic toxicity. However, OXAL induces neuropathic pain which hampers the chemotherapy success. Attempts with neuroprotective agents including anticonvulsivants and antidepressants were made to prevent OXAL‐induced painful neuropathy but the clinical data are controversial and the tested neuroprotectors are able to evoke themselves undesirable effects. Here, we demonstrated that the natural neurosteroid allopregnanolone (3&agr;,5&agr;‐THP), known to be devoid of toxic side‐effects in humans and experimental models, prevented and suppressed OXAL‐induced painful neuropathic symptoms. Indeed, 3&agr;,5&agr;‐THP repaired OXAL‐evoked neurochemical and functional alterations in peripheral nerves and intra‐epidermal nerve fibers (IENF). Behavioral analyses showed that prophylactic or corrective 3&agr;,5&agr;‐THP treatment (4 mg/kg/2 days) respectively prevented or abolished OXAL‐induced cold allodynia, mechanical allodynia and hyperalgesia by reversing to normal decreased thermal and mechanical pain thresholds of OXAL‐treated rats. Electrophysiological investigations revealed that 3&agr;,5&agr;‐THP restored control values of sciatic nerve conduction velocity and action potential peak amplitude drastically reduced by OXAL‐treatment. Furthermore, immunohistochemistry and confocal microscopic quantifications demonstrated that 3&agr;,5&agr;‐THP repaired OXAL‐induced neurochemical/cellular alterations by restoring IENF control density and normal level of neurofilament 200 kDa that was strongly repressed by OXAL in dorsal root ganglion neurons and sciatic nerve axons. OXAL showed no toxicity for the non‐compact myelin protein 2′,3′‐cyclic‐nucleotide‐3′‐phosphodiesterase whose expression level was similarly increased by 3&agr;,5&agr;‐THP in controls and OXAL‐treated rat nerves. Together, these results may be interesting for the development of natural or safe neurosteroid‐based neuroprotective strategy against anticancer drug‐evoked painful neuropathy.

Sciatic nerve injury induces apoptosis of dorsal root ganglion satellite glial cells and selectively modifies neurosteroidogenesis in sensory neurons

Neurosteroids are synthesized either by glial cells, by neurons, or within the context of neuron‐glia cross‐talk. Various studies suggested neurosteroid involvement in the control of neurodegeneration but there is no evidence showing that the natural protection of nerve cells against apoptosis directly depends on their own capacity to produce neuroprotective neurosteroids. Here, we investigated the interactions between neurosteroidogenesis and apoptosis occurring in sensory structures of rats subjected to neuropathic pain generated by sciatic nerve chronic constriction injury (CCI). Using the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL), we observed no apoptotic cells in the spinal cord up to 30 days after CCI although pain symptoms such as mechano‐allodynia, thermal and mechanical hyperalgesia were evidenced with the Hargreavess behavioral and von Frey filament tests. In contrast, double‐labeling experiments combining TUNEL and immunostaining with antibodies against glutamine synthetase or neuronal nuclei protein revealed apoptosis occurrence in satellite glial cells (SGC) (not in neurons) of CCI rat ipsilateral dorsal root ganglia (DRG) at day 30 after injury. Pulse‐chase experiments coupled with high performance liquid chromatography and flow scintillation detection showed that, among numerous biosynthetic pathways converting [3H]pregnenolone into various [3H]neurosteroids, only [3H]estradiol formation was selectively modified and upregulated in DRG of CCI rats. Consistently, immunohistochemical investigations localized aromatase (estradiol‐synthesizing enzyme) in DRG neurons but not in SGC. Pharmacological inhibition of aromatase caused apoptosis of CCI rat DRG neurons. Altogether, our results suggest that endogenously produced neurosteroids such as estradiol may be pivotal for the protection of DRG sensory neurons against sciatic nerve CCI‐induced apoptosis.

Improvement of neuronal bioenergetics by neurosteroids: Implications for age-related neurodegenerative disorders

The brain has high energy requirements to maintain neuronal activity. Consequently impaired mitochondrial function will lead to disease. Normal aging is associated with several alterations in neurosteroid production and secretion. Decreases in neurosteroid levels might contribute to brain aging and loss of important nervous functions, such as memory. Up to now, extensive studies only focused on estradiol as a promising neurosteroid compound that is able to ameliorate cellular bioenergetics, while the effects of other steroids on brain mitochondria are poorly understood or not investigated at all. Thus, we aimed to characterize the bioenergetic modulating profile of a panel of seven structurally diverse neurosteroids (progesterone, estradiol, estrone, testosterone, 3α-androstanediol, DHEA and allopregnanolone), known to be involved in brain function regulation. Of note, most of the steroids tested were able to improve bioenergetic activity in neuronal cells by increasing ATP levels, mitochondrial membrane potential and basal mitochondrial respiration. In parallel, they modulated redox homeostasis by increasing antioxidant activity, probably as a compensatory mechanism to a slight enhancement of ROS which might result from the rise in oxygen consumption. Thereby, neurosteroids appeared to act via their corresponding receptors and exhibited specific bioenergetic profiles. Taken together, our results indicate that the ability to boost mitochondria is not unique to estradiol, but seems to be a rather common mechanism of different steroids in the brain. Thus, neurosteroids may act upon neuronal bioenergetics in a delicate balance and an age-related steroid disturbance might be involved in mitochondrial dysfunction underlying neurodegenerative disorders.

Progress in dorsal root ganglion neurosteroidogenic activity: Basic evidence and pathophysiological correlation

Dorsal root ganglia (DRG) which contain glial cells and somas of primary sensory neurons are pivotal for neural transmission between the peripheral and central nervous systems. It is well established that neuropeptides such as substance P and calcitonin gene-related peptide located in DRG neurons control sensory and pain mechanisms. However, contrary to the brain and spinal cord which are extensively investigated, DRG received little attention. Therefore, the current knowledge on DRG may be far to represent their complete neurochemical potential. For instance, until 1997, nothing was known on DRG neurosteroidogenic ability but recently, several investigations have shown that DRG contain various key enzymes synthesizing neuroactive neurosteroids. To provide new advances into DRG neurochemistry, we reviewed and highlighted herein basic and functional evidence showing that neurosteroids are produced in DRG through a neuron-glia crosstalk mechanism. Indeed, key enzymes producing neurosteroids including pregnenolone, progesterone, dihydroprogesterone and estradiol are differentially expressed in DRG cell types. Cytochrome P450side-chain-cleavage is located in DRG neurons and satellite glial cells, 3beta-hydroxysteroid dehydrogenase is expressed in Schwann cells and neurons, 5alpha-reductase is localized in satellite glial and Schwann cells (not in neurons) while aromatase is present in neurons but not in glia. Recent studies also revealed that DRG neurosteroidogenesis is a physiologically relevant process selectively regulated under pathological conditions. Acting through paracrine and autocrine mechanisms, endogenous neurosteroids modulate DRG sensory functions and protect DRG neurons against death. The paper suggests that DRG neurosteroidogenic components may be targeted for the development of therapies against peripheral nerve injury-induced afferent noxious stimulations.

The neuroprotector kynurenic acid increases neuronal cell survival through neprilysin induction

Kynurenic acid (KYNA), one of the main product of the kynurenine pathway originating from tryptophan, is considered to be neuroprotective. Dysregulation of KYNA activity is thought to be involved in neurodegenerative diseases, the physiopathology of which evokes excitotoxicity, oxidative stress and/or protein aggregation. The neuroprotective effect of KYNA is generally attributed to its antagonistic action on NMDA receptors. However, this single target action appears insufficient to support KYNA beneficial effects against complex neurodegenerative processes including neuroinflammation, β-amyloid peptide (Aβ) toxicity and apoptosis. Novel insights are therefore required to elucidate KYNA neuroprotective mechanisms. Here, we combined cellular, biochemical, molecular and pharmacological approaches to demonstrate that low micromolar concentrations of KYNA strongly induce neprilysin (NEP) gene expression, protein level and enzymatic activity increase in human neuroblastoma SH-SY5Y cells. Furthermore, our studies revealed that KYNA exerts a protective effect on SH-SY5Y cells by increasing their viability through a mechanism independent from NMDA receptors. Interestingly, KYNA also induced NEP activity and neuroprotection in mouse cortical neuron cultures the viability of which was more promoted than SH-SY5Y cell survival under KYNA treatment. KYNA-evoked neuroprotection disappeared in the presence of thiorphan, an inhibitor of NEP activity. NEP is a well characterized metallopeptidase whose deregulation leads to cerebral Aβ accumulation and neuronal death in Alzheimers disease. Therefore, our results suggest that a part of the neuroprotective role of KYNA may depend on its ability to induce the expression and/or activity of the amyloid-degrading enzyme NEP in nerve cells.

Alzheimer, mitochondria and gender.

Epidemiological studies revealed that two-thirds of Alzheimers disease (AD) patients are women and the drop of sex steroid hormones after the menopause has been proposed to be one risk factor in AD. Similarly, the decrease of circulating testosterone levels with aging may also increase the risk of AD in men. Studies attest the neuroprotective effects of sex hormones in animal models of AD, but clinical trial data remain controversial. Here, we discuss the implication of mitochondria in gender differences observed in AD patients and animal models of AD. We summarize the role of mitochondria in aging and AD, pointing to the potential correlation between the loss of sex hormones and changes in the brain redox status. We discuss the protective effects of the sex hormones, estradiol, progesterone and testosterone with a specific focus on mitochondrial dysfunction in AD. The understanding of pathological processes linking the loss of sex hormones with mitochondrial dysfunction and mechanisms that initiate the disease onset may open new avenues for the development of gender-specific therapeutic approaches.

Selective regulation of 3α-hydroxysteroid oxido-reductase expression in dorsal root ganglion neurons: A possible mechanism to cope with peripheral nerve injury-induced chronic pain

&NA; The enzyme 3&agr;‐hydroxysteroid oxido‐reductase (3&agr;‐HSOR) catalyzes the synthesis and bioavailability of 3&agr;,5&agr;‐neurosteroids as allopregnanolone (3&agr;,5&agr;‐THP) which activates GABAA receptors and blocks T‐type calcium channels involved in pain mechanisms. Here, we used a multidisciplinary approach to demonstrate that 3&agr;‐HSOR is a cellular target the modulation of which in dorsal root ganglia (DRG) may contribute to suppress pain resulting from peripheral nerve injury. Immunohistochemistry and confocal microscope analyses showed 3&agr;‐HSOR‐immunostaining in naive rat DRG sensory neurons and glial cells. Pulse‐chase, high performance liquid chromatography and Flo/One characterization of neurosteroids demonstrated 3&agr;,5&agr;‐THP production in DRG. Behavioral methods allowed identification of pain symptoms (thermal and mechanical hyperalgesia and/or allodynia) in rats subjected to sciatic nerve chronic constriction injury (CCI). Reverse transcription and real‐time polymerase chain reaction revealed that 3&agr;‐HSOR mRNA concentration in CCI‐rat ipsilateral DRG, 5‐fold higher than in contralateral DRG, was also 4‐ to 6‐fold elevated than that in sham‐operated or naive rat DRG. Consistently, Western blotting confirmed increased 3&agr;‐HSOR protein levels in CCI‐rat ipsilateral DRG and double immunolabeling showed that 3&agr;‐HSOR overexpression occurred in DRG neurons but not in glia. Functional plasticity of 3&agr;‐HSOR leading to increased 3&agr;,5&agr;‐THP production was evidenced in CCI‐rat DRG. Interestingly, behavioral and molecular time‐course investigations revealed that 3&agr;‐HSOR gene upregulation was correlated to pain symptom development. Most importantly, in vivo knockdown of 3&agr;‐HSOR expression in healthy rat DRG using 6‐carboxyfluorescein‐3&agr;‐HSOR‐siRNA exacerbated thermal and mechanical pain perceptions. This paper is the first to show that siRNA‐induced knockdown of a key neurosteroid‐synthesizing enzyme directly affects an important function as nociception. Hopefully, these results may be useful for the development of novel analgesics.

Detecting spatial memory deficits beyond blindness in tg2576 Alzheimer mice

The retinal degeneration Pde6b(rd1) (rd) mutation can be a major pitfall in behavioral studies using tg2576 mice bred on a B6:SJL genetic background, 1 of the most widely used models of Alzheimers disease. After a pilot study in wild type mice, performance of 8- and 16-month-old tg2576 mice were assessed in several behavioral tasks with the challenge of selecting 1 or more task(s) showing robust memory deficits on this genetic background. Water maze acquisition was impossible in rd homozygotes, whereas Y-maze alternation, object recognition, and olfactory discrimination were unaffected by both the transgene and the rd mutation. Spatial memory retention of 8- and 16-month-old tg2576 mice, however, was dramatically affected independently of the rd mutation when mice had to recognize a spatial configuration of objects or to perform the Barnes maze. Thus, the latter tasks appear extremely useful to evaluate spatial memory deficits and to test cognitive therapies in tg2576 mice and other mouse models bred on a background susceptible to visual impairment.