Neurosteroidogenic Enzymes Research Articles

Pathogen odors elicit distinct neurosteroid and behavioral responses in Rainbow trout (Oncorhynchus mykiss)

Rainbow trout can smell virus and bacteria cues that in turn activate an immune response in the brain in absence of a systemic response (i.e. the immune response is activated by the nervous system instead of the pathogen inside tissues or bloodstream). However, little is known if pathogen odors can elicit different physiological responses (i.e. behavioral and immune) and how are pathogen odors integrated into different responses in the brain. We hypothesize that pathogen odors not only activate immune responses but also induce avoidance behavior. Moreover, we hypothesized that pathogen olfactory responses are modulated by neurosteroids at different times in different parts of the brain. We used a two-choice maze test to determine whether inactivated Yersinia ruckeri elicits aversive behavior. Furthermore, to check if behavioral and immune responses are modulated by neurosteroids, we performed a second experiment where we nasally administered inactivated Y. ruckeri to rainbow trout. After that, the nose, olfactory bulb, cerebellum, and optic tectum were collected at four different time points (15 min, 4 hours, 24 hours, and 7 days) and samples were extracted to measure gene expression of several steroidogenic enzymes ( cyp19a1, 3βHSD, 11HSD, and 17HSD) and fish production of neurosteroids (e.g. cortisol, estradiol, and progesterone). We found that rainbow trout actively avoids pathogen odors. Moreover, we found a different expression of neuro steroidogenic enzymes at different brain areas and times post-exposure. Fish exposed to pathogen odor express a significant increase (ANOVA 2 factor, P<0.05) of cyp19a1 (involved in estradiol synthesis) as soon as 15 min in the nose and olfactory bulb, and 4 hours post-exposure in the cerebellum and optic tectum. This gene expression was parallel to a significant increase of estradiol in the nose, bulb, and rest of the brain between 15 min and 4 hours. Moreover, there was a significant increase of 11HSD and 17HSD (related to the production of testosterone, cortisol, and corticosterone) 4 hours after exposure in the nose and cerebellum but a significant decrease in the olfactory bulb and optic tectum. These gene expression patterns were parallel o an increase of cortisol in the nose and olfactory bulb. Finally, we found a significant increase of 3βHSD (involved in the first steps of steroidogenesis) after 24 hours in the nose and olfactory bulb, although we didn’t find an increase in steroid production in our preliminary assays. Our results showed that exposure to pathogen odors activates different neuro steroidogenic pathways at different times after exposure, and at different areas of the brain. These findings pinpoint the use of the sense of smell by fish to control behavioral and immune responses to pathogens, with neurosteroids playing a key role in modulating and integrating these olfactory responses. This material is based upon work supported by the National Science Foundation under Grant No. 1755348 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prenatal Stress Induces Translational Disruption Associated with Myelination Deficits

Disruptions to neurodevelopment are known to be linked to behavioral disorders in childhood and into adulthood. The fetal brain is extremely vulnerable to stimuli that alter inhibitory GABAergic pathways and critical myelination processes, programing long-term neurobehavioral disruption. The maturation of the GABAergic system into the major inhibitory pathway in the brain and the development of oligodendrocytes into mature cells capable of producing myelin are integral components of optimal neurodevelopment. The current study aimed to elucidate prenatal stress-induced mechanisms that disrupt these processes and to delineate the role of placental pathways in these adverse outcomes. Pregnant guinea pig dams were exposed to prenatal stress with strobe light exposure for 2 h/day on gestational age (GA) 35, 40, 45, 50, 55, 60, and 65, and groups of fetuses and placentae were collected after the stress exposure on GA40, GA50, GA60, and GA69 (term). Fetal plasma, placental, and brain tissue were collected for allopregnanolone and cortisol quantification with ELISA. Relative mRNA expression of genes of specific pathways of interest was examined with real-time PCR in placental and hippocampal tissue, and myelin basic protein (MBP) was quantified immunohistochemically in the hippocampus and surrounding regions for assessment of mature myelin. Prenatal stress in mid-late gestation resulted in disruptions to the translational machinery responsible for the production of myelin and decreased myelin coverage in the hippocampus and surrounding regions. The male placenta showed an initial protective increase in allopregnanolone concentrations in response to maternal psychosocial stress. The male and female placentae had a sex-dependent increase in neurosteroidogenic enzymes at term following prenatal stress. Independent from exposure to prenatal stress, at gestational day 60 – a critical period for myelin development, the placentae of female fetuses had increased capability of preventing cortisol transfer to the fetus through expression of 11-beta-hydroxysteroid dehydrogenase types 1 and 2. The deficits early in the process of maturation of myelination indicate that the reduced myelination observed at childhood equivalence in previous studies begins in fetal life. This negative programing persists into childhood, potentially due to dysregulation of MBP translation processes. Expression patterns of neurosteroidogenic enzymes in the placenta at term following stress may identify at-risk fetuses that have been exposed to a stressful in utero environment.

Taxifolin Inhibits Neurosteroidogenic Rat Steroid 5α-Reductase 1 and 3α-Hydroxysteroid Dehydrogenase

Aims: Taxifolin, a flavonoid, has several pharmacological activities. Taxifolin inhibits some steroid biosynthetic enzymes, suggesting that it might inhibit neurosteroid synthesis. Here, we investigated effects of taxifolin on rat neurosteroidogenic enzymes, steroid 5α-reductase 1 ­(SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C9), and retinol dehydrogenase 2 (RDH2). Methods: SRD5A1, AKR1C9, and RDH2 were expressed in COS-1 cells and the direct effects of taxifolin on these enzymes and the mode of action were determined using steroid substrates and thin chromatography separation-coupled radiometric scanning. Results: The half maximal inhibitory concentration values of taxifolin on SRD5A1 and AKR1C9 were 100 and 1.01 mol/L. Taxifolin did not inhibit RDH2 even at as high as 100 mol/L. Taxifolin competitively inhibited rat AKR1C9 against steroid dihydrotestosterone (DHT), and docking analysis revealed that taxifolin bound to the steroid binding site of rat AKR1C9 with similar free energy with the substrate DHT. Conclusion: Taxifolin is a potent inhibitor of rat AKR1C9 and moderate inhibitor of rat SRD5A1, thereby controlling the rate of neurosteroid biosynthesis.

Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus.

Neurosteroids are potent allosteric modulators of GABAA receptors (GABAA Rs). Although the effects of exogenous neurosteroids on GABAA R function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABAA R-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABAA R function by 5α-reduced neurosteroids in the mature thalamus.

Inhibition of Cytochrome P450 Side-Chain Cleavage Attenuates the Development of Mechanical Allodynia by Reducing Spinal D-Serine Production in a Murine Model of Neuropathic Pain.

Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Spinal Interleukin-1β Inhibits Astrocyte Cytochrome P450c17 Expression Which Controls the Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain

We have recently demonstrated that sciatic nerve injury increases the expression of spinal cytochrome P450c17, a key neurosteroidogenic enzyme, which plays a critical role in the development of peripheral neuropathic pain. However, the modulatory mechanisms responsible for the expression of spinal P450c17 have yet to be examined. Here we investigated the possible involvement of interleukin-1β (IL-1β) in altering P450c17 expression during the induction phase of neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in mice and mechanical allodynia was evaluated in the hind paws using a von-Frey filament (0.16 g). Western blotting and immunohistochemistry were performed to assess the expression of spinal IL-1β, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1β was significantly increased on day 1 post-surgery and its receptor, IL-1R1 was expressed in GFAP-positive astrocytes. Intrathecal administration of the recombinant interleukin-1 receptor antagonist (IL-1ra, 20 ng) on days 0 and 1 post-surgery enhanced GFAP expression on day 1 post-surgery and induced an early increase in P450c17 expression in astrocytes, but not in neurons. Administration of IL-1β (10 ng) on days 0 and 1 post-surgery blocked the enhancement of both spinal P450c17 and GFAP expression induced by IL-1ra (20 ng) administration. Intrathecal administration of IL-1ra (20 ng) on days 0 to 3 post-surgery also facilitated the CCI-induced development of mechanical allodynia, and this early developed pain was dose-dependently attenuated by the administration of the P450c17 inhibitor, ketoconazole (1, 3, or 10 nmol) or the astrocyte metabolic inhibitor, fluorocitrate (0.01, 0.03, or 0.1 nmol). These results demonstrate that early increases in spinal IL-1β temporally inhibit astrocyte P450c17 expression and astrocyte activation ultimately controlling the development of mechanical allodynia induced by peripheral nerve injury. These findings imply that spinal IL-1β plays an important role as an early, but transient, control mechanism in the development of peripheral neuropathic pain via the inhibition of astrocyte P450c17 expression and astrocyte activation.

Spinal cytochrome P450c17 plays a key role in the development of neuropathic mechanical allodynia: Involvement of astrocyte sigma-1 receptors

While evidence indicates that sigma-1 receptors (Sig-1Rs) play an important role in the induction of peripheral neuropathic pain, there is limited understanding of the role that the neurosteroidogenic enzymes, which produce Sig-1R endogenous ligands, play during the development of neuropathic pain. We examined whether sciatic nerve injury upregulates the neurosteroidogenic enzymes, cytochrome P450c17 and 3β-hydroxysteroid dehydrogenase (3β-HSD), which modulate the expression and/or activation of Sig-1Rs leading to the development of peripheral neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of P450c17, but not 3β-HSD, in the ipsilateral lumbar spinal cord dorsal horn at postoperative day 3. Intrathecal administration of the P450c17 inhibitor, ketoconazole during the induction phase of neuropathic pain (day 0 to day 3 post-surgery) significantly reduced the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw. However, administration of the 3β-HSD inhibitor, trilostane had no effect on the development of neuropathic pain. Sciatic nerve injury increased astrocyte Sig-1R expression as well as dissociation of Sig-1Rs from BiP in the spinal cord. These increases were suppressed by administration of ketoconazole, but not by administration of trilostane. Co-administration of the Sig-1R agonist, PRE084 restored the development of mechanical allodynia originally suppressed by the ketoconazole administration. However, ketoconazole-induced inhibition of thermal hyperalgesia was not affected by co-administration of PRE084. Collectively these results demonstrate that early activation of P450c17 modulates the expression and activation of astrocyte Sig-1Rs, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Stimulation of Peroxisome Proliferator-Activated Receptor-α by N-Palmitoylethanolamine Engages Allopregnanolone Biosynthesis to Modulate Emotional Behavior

BackgroundThe endocannabinoid and neurosteroid systems regulate emotions and stress responses. Activation of peroxisome proliferator-activated receptor (PPAR)-α by the endocannabinoid congener N-palmitoylethanolamine (PEA) regulates pathophysiological systems (e.g., inflammation, oxidative stress) and induces peripheral biosynthesis of allopregnanolone, a gamma-aminobutyric acidergic neurosteroid implicated in mood disorders. However, effects of PPAR-α on emotional behavior are poorly understood. MethodsWe studied the impact of PPAR-α activation on emotional behavior in a mouse model of posttraumatic stress disorder. Neurosteroid levels before and after PEA treatment were measured by gas chromatography–mass spectrometry in relevant brain regions of socially isolated versus group-housed mice exposed to the contextual fear conditioning test, elevated plus maze test, forced swim test, and tail suspension test. Neurosteroidogenic enzyme levels were quantified in hippocampus by Western blot. ResultsPEA administered in a model of conditioned contextual fear reconsolidation blockade facilitated fear extinction and fear extinction retention and induced marked antidepressive- and anxiolytic-like effects in socially isolated mice with reduced brain allopregnanolone levels. These effects were mimicked by the PPAR-α synthetic agonists, fenofibrate and GW7647, and were prevented by PPAR-α deletion, PPAR-α antagonists, and neurosteroid-enzyme inhibitors. Behavioral improvements correlated with PEA-induced upregulation of PPAR-α, neurosteroidogenic enzyme expression, and normalization of corticolimbic allopregnanolone levels. ConclusionsThis evidence supports a previously unknown role for PPAR-α in behavior regulation and suggests new strategies for the treatment of neuropsychopathologies characterized by deficient neurosteroidogenesis, including posttraumatic stress disorder and major depressive disorder.

The impact of sensory and motor enrichment on the epigenetic control of steroidogenic-related genes in rat hippocampus

In the present study, we analyzed the effects of a short-term environmental enrichment on the mRNA expression and DNA methylation of steroidogenic enzymes in the hippocampus. Thus, young adult (80-day-old) and middle-aged (350-day-old) Wistar female rats were exposed to sensory (SE) or motor (ME) enrichment during 10 days and compared to animals housed under standard conditions. SE was provided by an assortment of objects that included plastic tubes and toys; for ME, rodent wheels were provided. In young adult animals, SE and ME increased the mRNA expression of cytochrome P450 17α-hydroxylase/c17,20-lyase, steroid 5α-reductase type 1 (5αR-1) and 3α-hydroxysteroid dehydrogenase and decreased the methylation levels of 5αR-1 gene. In middle-aged rats, ME and SE upregulated the gene expression of aldosterone synthase and decreased the methylation state of its promoter. These results propose that SE and ME differentially regulate the transcription of neurosteroidogenic enzymes through epigenetic mechanisms in young and aged rats.

Changes in neurosteroidogenesis during demyelination and remyelination in cuprizone-treated mice.

Changes of neurosteroids may be involved in the pathophysiology of multiple sclerosis (MS). The present study investigated whether changes of neurosteroidogenesis also occurred in the grey and white matter regions of the brain in mice subjected to cuprizone-induced demyelination. Accordingly, we compared the expression of neurosteroidogenic proteins, including steroidogenic acute regulatory protein (StAR), voltage-dependent anion channel (VDAC) and 18 kDa translocator protein (TSPO), as well as neurosteroidogenic enzymes, including the side chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase/isomerase and 5α-reductase (5α-R), during the demyelination and remyelination periods. Using immunohistochemistry and a quantitative polymerase chain reaction, we demonstrated a decreased expression of StAR, P450scc and 5α-R with respect to an increase astrocytic and microglial reaction and elevated levels of tumor necrosis factor (TNF)α during the cuprizone demyelination period in the hippocampus, cortex and corpus callosum. These parameters, as well as the glial reaction, were normalised after 2weeks of spontaneous remyelination in regions containing grey matter. Conversely, persistent elevated levels of TNFα and low levels of StAR and P450scc were observed during remyelination in corpus callosum white matter. We conclude that neurosteroidogenesis/myelination status and glial reactivity are inversely related in the hippocampus and neocortex. Establishing a cause and effect relationship for the measured variables remains a future challenge for understanding the pathophysiology of MS.

Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.

Background: Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABA AR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABA AR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods: Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy ( ob/ob) and WT controls aged 60-80 days. Results: There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions: The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABA AR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathy, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.

Neurosteroidogenesis and progesterone anti-inflammatory/neuroprotective effects.

Progesterone shows anti-inflammatory and promyelinating effects in mice with experimental autoimmune encephalomyelitis (EAE), a commonly used model for multiple sclerosis (MS). Because neurosteroids have been implicated as protective factors for MS and EAE, we analysed the expression of neurosteroidogenic enzymes in the compromised spinal cord of EAE mice. EAE was induced in female C57Bl6 mice, which were then killed on day 16 after induction. Progesterone was given by pellet implantation 1week before EAE induction. Untreated EAE mice showed decreased mRNAs for the steroidogenic acute regulatory protein (Star), voltage-dependent anion channel (VDAC), cholesterol side-chain cleavage (P450scc), 5α-reductase, 3α-hydroxysteroid dehydrogenase (3α-HSOR) and aromatase, whereas changes of 3β-hydroxysteroid dehydrogenase (3β-HSD) were not significant. mRNA translocator protein (18kDa) (TSPO) was elevated, concomitantly with a reactive microgliosis. EAE mice also showed abnormal mitochondrial ultrastructure in axons and neuronal bodies, as well as reduced expression of fission and fusion protein mRNAs. Progesterone pretreatment before EAE induction increased Star, VDAC, P450scc, 5α-reductase type I, 3α-HSOR and aromatase mRNAs and did not modify 3β-HSD. TSPO mRNA was decreased, possibly as a result of reversal of microgliosis. Progesterone pretreatment also improved mitochondrial ultrastructure and increased fission/fusion protein mRNAs. These mitochondrial effects may be part of the progesterone recovery of neurosteroidogenesis. The enzymes 3β-HSD, 3α-HSOR and 5α-reductase are also responsible for the formation of androgens. Because MS patients and EAE rodents show changes of central androgen levels, it is likely that, together with progestins and oestrogens, neuroandrogens afford neuroprotection for EAE and MS. The data reviewed suggest that enhanced synthesis of neurosteroids contributes in an auto/paracrine manner to reinforce the neuroprotective and anti-inflammatory effects of exogenous progesterone given to EAE mice.

Sex hormone levels in the brain of d-aspartate-treated rats

d-Aspartate (d-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. d-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days d-Asp drinking solution) and acute (i.p. injection of 2μmol/g bw d-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of d-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of d-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that d-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30min after d-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29–35%, and at 8h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of d-Asp to brain homogenate+substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.

Apigenin inhibits rat neurosteroidogenic 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase

Apigenin, a common flavonoid, has extensive pharmacological activities. Apigenin inhibits some steroid biosynthetic enzymes, suggesting that it may block neurosteroid synthesis. Neurosteroids play many important roles in neurological functions. The objective of the present study is to investigate effects of apigenin on neurosteroidogenic enzymes, 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C9), and retinol dehydrogenase 2 (RoDH2), in rats. SRD5A1, AKR1C9, and RoDH2 were expressed in COS-1 cells and the effects of apigenin on these enzymes and modes of action were explored using radiolabeled substrates and thin-layer chromatographic separation coupled with radiometry. Apigenin inhibited SRD5A1, AKR1C9, and RoDH2 activities with IC50 values of 100, 0.891 ± 0.065, and >100 μM, respectively. Apigenin competitively inhibited rat AKR1C9 when its substrate 5α-dihydrotestosterone was used and uncompetitively inhibited the enzyme when cofactor NADPH was used. In conclusion, apigenin is a potent inhibitor of rat AKR1C9, thereby controlling the rate of neurosteroid biosynthesis.

Effects of resveratrol on rat neurosteroid synthetic enzymes

Resveratrol, a common polyphenol, has extensive pharmacological activities. Resveratrol inhibits some steroid biosynthetic enzymes, indicating that it may block neurosteroid synthesis. The objective of the present study is to investigate the inhibition of resveratrol on neurosteroidogenic enzymes rat 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C9), and retinol dehydrogenase 2 (RDH2). The IC50 values of resveratrol on SRD5A1, AKR1C9, and RDH2 were >100μM, 0.436±0.070μM, and 4.889±0.062μM, respectively. Resveratrol competitively inhibited rat AKR1C9 and RDH2 against steroid substrates. Docking showed that resveratrol bound to the steroid binding pocket of AKR1C9. It exerted a mixed mode on these AKR1C9 and RDH2 against cofactors. In conclusion, resveratrol potently inhibited rat AKR1C9 and RDH2 to regulate local neurosteroid levels.

Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.

Background : Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABA AR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABA AR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods : Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy ( ob/ob) and WT controls aged 60-80 days. Results : There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions : The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABA AR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathy, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.

Cafeteria diet differentially alters the expression of feeding-related genes through DNA methylation mechanisms in individual hypothalamic nuclei

We evaluated the effect of cafeteria diet (CAF) on the mRNA levels and DNA methylation state of feeding-related neuropeptides, and neurosteroidogenic enzymes in discrete hypothalamic nuclei. Besides, the expression of steroid hormone receptors was analyzed. Female rats fed with CAF from weaning increased their energy intake, body weight, and fat depots, but did not develop metabolic syndrome. The increase in energy intake was related to an orexigenic signal of paraventricular (PVN) and ventromedial (VMN) nuclei, given principally by upregulation of AgRP and NPY. This was mildly counteracted by the arcuate nucleus, with decreased AgRP expression and increased POMC and kisspeptin expression. CAF altered the transcription of neurosteroidogenic enzymes in PVN and VMN, and epigenetic mechanisms associated with differential promoter methylation were involved. The changes observed in the hypothalamic nuclei studied could add information about their differential role in food intake control and how their action is disrupted in obesity.

Mitochondrial dysfunction in diabetic neuropathy may be involved in the development of neuropathic pain via a reduction in neurosteroid synthesis

Background: Recent work in a model of diabetic neuropathy revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABAAR and exogenously applied neurosteroids had an exaggerated impact. It is postulated that this is related to reduced precursor synthesis, due to mitochondrial dysfunction in diabetic neuropathy. Benzodiazepines are also known to activate neurosteroidogenesis by binding to mitochondrial translocator protein (TSPO). This study explored the differential effect of diazepam on GABAAR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Methods: Whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from layer 2/3 cortical pyramidal neurons of the pain pathway from mice with type-II diabetic neuropathy (ob/ob) and WT controls aged 60-80 days. Results: There was a key difference in the response of the WT and ob/ob cortical neurons to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5a-reductase inhibitor finasteride, individually or in combination, produced the same response in both strains. Conclusions: The exaggerated effect of diazepam on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABAAR benzodiazepine antagonist flumazenil is likely observed due to physiological upregulation of key neurosteroidogenic enzymes in response to the reduced pregnenolone synthesis by the mitochondria. By increasing pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and increase GABAergic inhibitory tone via an alternate route. In diabetic neuropathic pain, mitochondrial dysfunction may play an important role. Enhancing the GABAergic neurosteroid tone could be of potential therapeutic benefit.

Effects of Fungicides on Rat's Neurosteroid Synthetic Enzymes.

Exposure to environmental endocrine disruptors may interfere with nervous system's activity. Fungicides such as tebuconazole, triadimefon, and vinclozolin have antifungal activities and are used to prevent fungal infections in agricultural plants. In the present study, we studied effects of tebuconazole, triadimefon, and vinclozolin on rat's neurosteroidogenic 5α-reductase 1 (5α-Red1), 3α-hydroxysteroid dehydrogenase (3α-HSD), and retinol dehydrogenase 2 (RDH2). Rat's 5α-Red1, 3α-HSD, and RDH2 were cloned and expressed in COS-1 cells, and effects of these fungicides on them were measured. Tebuconazole and triadimefon competitively inhibited 5α-Red1, with IC50 values of 8.670 ± 0.771 × 10−6 M and 17.390 ± 0.079 × 10−6 M, respectively, while vinclozolin did not inhibit the enzyme at 100 × 10−6 M. Triadimefon competitively inhibited 3α-HSD, with IC50 value of 26.493 ± 0.076 × 10−6 M. Tebuconazole and vinclozolin weakly inhibited 3α-HSD, with IC50 values about 100 × 10−6 M, while vinclozolin did not inhibit the enzyme even at 100 × 10−6 M. Tebuconazole and triadimefon weakly inhibited RDH2 with IC50 values over 100 × 10−6 M and vinclozolin did not inhibit this enzyme at 100 × 10−6 M. Docking study showed that tebuconazole, triadimefon, and vinclozolin bound to the steroid-binding pocket of 3α-HSD. In conclusion, triadimefon potently inhibited rat's neurosteroidogenic enzymes, 5α-Red1 and 3α-HSD.

Seasonal changes and sexual dimorphism in gene expression of StAR protein, steroidogenic enzymes and sex hormone receptors in the frog brain

The brain of amphibians contains all the key enzymes of steroidogenesis and has a high steroidogenic activity. In seasonally-breeding amphibian species brain steroid levels fluctuate synchronously with the reproductive cycle. Here we report a study of gene expression of StAR protein, key steroidogenic enzymes and sex hormone receptors in the telencephalon (T) and diencephalon-mesencephalon (D-M) of male and female reproductive and post-reproductive Pelophylax esculentus, a seasonally breeding anuran amphibian. Significant differences in gene expression were observed between (a) the reproductive and post-reproductive phase, (b) the two brain regions and (c) male and female frogs. During the reproductive phase, star gene expression increased in the male (both T and D-M) but not in the female brain. Seasonal fluctuations in expression levels of hsd3b1, hsd17b1, srd5a1 and cyp19a1 genes for neurosteroidogenic enzymes occurred in D-M region of both sexes, with the higher levels in reproductive period. Moreover, the D-M region generally showed higher levels of gene expression than the T region in both sexes. Gene expression was higher in females than males for most genes, suggesting higher neurosteroid production in female brain. Seasonal and sex-linked changes were also observed in gene expression for androgen (ar) and estrogen (esr1, esr2) receptors, with the males showing the highest ar levels in reproductive phase and the highest esr1 and esr2 levels in post-reproductive phase; in contrast, females showed the maximum expression for all three genes in reproductive phase. The results are the first evidence for seasonal changes and sexual dimorphism of gene expression of the neurosteroidogenic pathway in amphibians.