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Neuroprotective effects of allopregnenolone on hippocampal irreversible neurotoxicity in vitro
Abstract
1.1. The effects of allopregnenolone, a neurosteroid, endowed with GABAmimetic properties, were tested towards two models of irreversible hippocampal neurotoxicity: i) the irreversible depression produced by hypoxia on the CA1 evoked field potentials in rat hippocampal slices, and ii) glutamateinduced irreversible changes in intracellular calcium concentration in primary hippocampal cell coltures.2.2. In control conditions during the reoxygenation period after the application of 15 min of hypoxia, the CA1 evoked field potentials were irreversibly suppressed in almost the 50% of the experiments. In the remaining experiments there were a significative (p < 0.01) irreversible reduction of the magnitude of the CA1 population spike with respect with the pre-hypoxia values. Allopregnenolone (50–75 μM) perfused 30 min before, during and 30 min after hypoxia produced a significative (p < 0.05) decrease both in the hypoxia-induced irreversible suppression of the CA1 PS and both in the irreversible decrease of the CA1 PS at the end of reoxygenation.3.3. The exposition of the primary hippocampal cultured cells to glutamate 0.5 mM for 10 min was followed by a sustained elevation of [Ca2+] i, that persisted at 70–80% of maximal increase for the rest of the experiment (60 min). When a pretreatment with 10–50 μM allopregnanolone preceded Glu 0.5 mM application, [Ca2+] i increased to a maximal value during the glutamate application, after which a fast decrease to 50% was observed, followed by a slow recovery within about 30 min.4.4. The results showed that the neurosteroid allopregnenolone, endowed with GABAmimetic properties, ameliorated the functional correlates of irreversible hippocampal neurotoxicity.
... Carbamazepine has also been reported to increase the concentrations of allopregnanolone, pregnenolone, progesterone and THDOC in the cerebral cortex as well as in plasma [16], perhaps by stimulating the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) [13,16]. The allopregnanolone and pregnenolone has been documented to produce a neuroprotective effect [31,32] by activating GABA receptors [33] and by inhibiting glutamate induced irreversible changes in the intracellular Ca 2+ concentration [34]. Therefore, it may be possible to speculate that carbamazepine induced attenuation of ischaemia-reperfusion-induced increase in cerebral infarct size noted in the present study is due to increase in synthesis of neurosteroids. ...
... Carbamazepine has attenuated ischaemia-reperfusion-induced increase in TBARS in mitochondria and cytoplasm, which is the index of lipid peroxidation. This may be due to the fact that carbamazepine has increased the synthesis of neurosteroids which in turn may have reduced glutamate induced Ca 2+ overload [34] and hence has decreased lipid peroxidation and TBARS. This is further supported by the observation that indomethacin, an inhibitor of 3α-HSD, has attenuated carbamazepine induced decrease in TBARS. ...
... Brain tissue infarction as a result of oxygen free radicals has been noted to decrease motor performance and motor co-ordination. The increase in neurosteroid synthesis due to activation of 3α-HSD and the consequent prevention of generation of reactive oxygen species [34] may be responsible for the noted ameliorative effect of carbamazepine on ischaemia-reperfusion-induced impairment of motor performance. This point is further supported by our observation with indomethacin, an inhibitor of 3α-HSD, who attenuated the neuroprotective effect of carbamazepine on motor performance. ...
The present study was designed to investigate the putative effect of neurosteroid modulation on global ischaemia-reperfusion-induced cerebral injury in mice. Bilateral carotid artery occlusion followed by reperfusion, produced a significant rise in cerebral infarct size along with impairment of grip strength and motor coordination in Swiss albino mice. Administration of carbamazepine (16 mg/kg, i.p.) before global cerebral ischaemia significantly attenuated cerebral infarct size and improved the motor performance. However, administration of indomethacin (100 mg/kg, i.p.) attenuated the neuroprotective effect of carbamazepine. Mexiletine (50 mg/kg, i.p.) did not produce significant neuroprotective effect. It may be concluded that the neuroprotective effect of carbamazepine may be due to increase in synthesis of neurosteroids perhaps by activating enzyme (3α HSD) as indomethacin attenuated the neuroprotective effect of carbamazepine. The sodium channel blocking effect of carbamazepine may not be involved in neuroprotection as mexiletine, a sodium channel blocker, did not produce significant neuroprotective effect.
... Treatment with allopregnanolone protects against cell death in adult rat models of traumatic brain injury, (He et al., 2004a;Djebaili et al., 2005), reduces excitotoxicity in vitro (Frank and Sagratella, 2000), has anti-seizure effects (Frye, 1995), and anti-inflammatory effects (He et al., 2004b). Allopregnanolone has also been demonstrated to cross the placenta, reducing effects of prenatal stress on fetuses, thereby reducing adolescent anxiety in rats (Zimmerberg and Blaskey, 1998) DHP is then reversibly converted to THP (allotetrahydroprogesterone/Allopregnanolone) via 3α-HSD (3α-hydroxysteroid dehydrogenase). ...
... Allopregnanolone is a 3α, 5α-reduced pregnane steroid produced de-novo within the brain (see, Belelli and Lambert, 2005). This endogenous neurosteroid regulates basal levels of apoptosis (Yawno et al., 2007); has anti-seizure (Frye, 1995) and anti-inflammatory effects (He et al., 2004b); and reduces cell death from hypoxia/ischemia, excitotoxicity, and traumatic brain injury (Frank and Sagratella, 2000;Djebaili et al., 2004;He et al., 2004a;Djebaili et al., 2005). ...
Perinatal brain injury is a major cause of morbidity and mortality in humans. Birth
asphyxia and pre-term birth cause patterns of brain damage that depend on sex, and
gestational age at the time of insult. Where asphyxia or prematurity cause brain damage the
hippocampus, an area brain critically important in learning and memory, is often affected.
Using spiny mice we have investigated the clinically relevant insult of late gestation
fetal hypoxia and metabolic acidosis on the neonatal hippocampus. We used
electrophysiological and immunohistological techniques to investigate synaptic function
hippocampal plasticity after birth asphyxia and pre-term birth. We also determined if an
endogenous neurosteroid, allopregnanolone prevent hippocampal dysfunction in the
context of birth asphyxia.
... We found that allopregnanolone inhibited the depolarization-evoked Ca 2+ -dependent exocytotic release of glutamate, and this phenomenon was observed to be dose-dependent and was maximal at 20 µM, with an IC 50 of 10 µM. The concentration of allopregnanolone we used is similar to previous studies, which have shown that 10 to 50 µM allopregnanolone inhibits glutamate-induced increases in intracellular Ca 2+ and neuronal death in cultured hippocampal neurons and human NT2 neurons (Frank & Sagratella, 2000;Lockhart et al., 2002). The mechanism(s) contributing to the allopregnanolone-mediated inhibition of glutamate release from cerebrocortical synaptosomes are discussed as follows. ...
... Second, allopregnanolone significantly reduced the evoked increase in intrasynaptosomal Ca 2+ levels. This finding is consistent with a report by Frank and Sagratella (2000), which demonstrated that allopregnanolone decreases glutamate-induced increases in intracellular Ca 2+ in rat hippocampal slices. Third, allopregnanolone did not affect the synaptosomal plasma membrane potential either in the resting state or after depolarization with 4-AP. ...
Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase (AC), and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABAA receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABAA receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.
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... The dose-dependent effects of Δ 5 PS on locomotion of mice placed in a novel environment show the existence of a possible role of neurosteroids in adaptation to novelty (Fahey et al., 1995a ). Neurosteroids are also involved in the regulation of excitotoxic and apoptotic processes (Kimonides et al., 1999; Frank and Sagratella, 2000; Manji et al., 2003; Charalampopoulos et al., 2004; Wojtal et al., 2006 ). In vivo studies indicate that progesterone, allopregnanolone , and DHEA exert neuroprotective effects in models of traumatic brain injury (Stein, 2001; Malik et al., 2003; Djebaili et al., 2004; He et al., 2004) and in focal cerebral ischemia (Malik et al., 2003; Sayeed et al., 2006). ...
... In vivo studies indicate that progesterone, allopregnanolone , and DHEA exert neuroprotective effects in models of traumatic brain injury (Stein, 2001; Malik et al., 2003; Djebaili et al., 2004; He et al., 2004) and in focal cerebral ischemia (Malik et al., 2003; Sayeed et al., 2006). In vitro data also point to the neuroprotective effects of neurosteroids in models of neuronal injury against neurotoxic insults inflicted by excitatory amino acids (Kimonides et al., 1999; Gursoy et al., 2001) and β-amyloid peptides (Kimonides et al., 1998Kimonides et al., , 1999 Frank and Sagratella, 2000; Lockhart et al., 2002; Xilouri and Papazafiri, 2006). It has been demonstrated that neurosteroids play important functions in neurodevelopment and neuronal remodeling, including neurogenesis (Roberts, 1986; Weaver et al., 1997; Schumacher et al., 2000; Magnaghi et al., 2001; Young, 2002), axonal and dendritic growth, and synaptic connectivity (Jones, 1994; Compagnone and Mellon, 1998). ...
The enzymatic pathways leading to the synthesis of bioactive steroids in the brain are now almost completely elucidated in various groups of vertebrates and, during the last decade, the neuronal mechanisms involved in the regulation of neurosteroid production have received increasing attention. This report reviews the current knowledge concerning the effects of neurotransmitters, peptide hormones, and neuropeptides on the biosynthesis of neurosteroids. Anatomical studies have been carried out to visualize the neurotransmitter- or neuropeptide-containing fibers contacting steroid-synthesizing neurons as well as the neurotransmitter, peptide hormones, or neuropeptide receptors expressed in these neurons. Biochemical experiments have been conducted to investigate the effects of neurotransmitters, peptide hormones, or neuropeptides on neurosteroid biosynthesis, and to characterize the type of receptors involved. Thus, it has been found that glutamate, acting through kainate and/or AMPA receptors, rapidly inactivates P450arom, and that melatonin produced by the pineal gland and eye inhibits the biosynthesis of 7α-hydroxypregnenolone (7α-OH-Δ⁵P), while prolactin produced by the adenohypophysis enhances the formation of 7α-OH-Δ⁵P. It has also been demonstrated that the biosynthesis of neurosteroids is inhibited by GABA, acting through GABAA receptors, and neuropeptide Y, acting through Y1 receptors. In contrast, it has been shown that the octadecaneuropetide ODN, acting through central-type benzodiazepine receptors, the triakontatetraneuropeptide TTN, acting though peripheral-type benzodiazepine receptors, and vasotocin, acting through V1a-like receptors, stimulate the production of neurosteroids. Since neurosteroids are implicated in the control of various neurophysiological and behavioral processes, these data suggest that some of the neurophysiological effects exerted by neurotransmitters and neuropeptides may be mediated via the regulation of neurosteroid production.
... Reports that allopregnanolone levels increase in the brain in response to stress, including hypoxic-ischemia (HI; Barbaccia et al., 1996;Nguyen et al., 2004), suggest that allopregnanolone may function as an endogenous neuroprotectant. Supporting this, inhibition of allopregnanolone synthesis increases cell death following HI in fetal sheep (Yawno et al., 2007), and treatment with allopregnanolone in vitro ameliorates HI induced neuronal loss (Frank and Sagratella, 2000), and reduces the size of brain lesion following adult traumatic brain injury (Djebaili et al., 2005). ...
... Furthermore, the single dose of this neurosteroid reduced proliferation in the dentate gyrus at P5. Hypoxic-ischemia commonly gives rise to brain injury, including decreased LTP (Dai et al., 2007;Svedin et al., 2007;Towfighi et al., 1997), due largely to the effects of excitotoxicity (Ikonomidou et al., 1989). The finding that allopregnanolone ameliorated some of these functional deficits in our model of perinatal HI is consistent with the ability of this neurosteroid to reduce glutamate neurotoxicity in vivo (Frank and Sagratella, 2000). This action likely involves increasing GABAergic activity and reduced pre-synaptic glutamate release by antagonising L-type calcium channels, thus reducing asphyxia induced excitation. ...
... The underlying mechanisms and the targets of their neuroprotective effects have not been elucidated. As the major active metabolite of PROG, ALLO has been shown to have neuroprotective properties both in vitro (Frank and Sagratella, 2000) and in vivo (Morali et al., 2011). The prevailing view holds that PROG exerts its neuroprotective effects through multiple receptors: classical progesterone receptors (Pgr), PGRMC1, membrane progesterone receptors (mPR), and GABA A receptors after conversion to ALLO (Cooke et al., 2013;Guennoun et al., 2015). ...
Ketamine exposure can model cognitive deficits associated with schizophrenia. Progesterone (PROG) and its active metabolite allopregnanolone (ALLO) have neuroprotective effects and the pathway involving progesterone receptor membrane component 1 (PGRMC1), epidermal growth factor receptor (EGFR), glucagon-like peptide-1 receptor (GLP-1R), phosphatidylinositol 3 kinase (PI3K), and protein kinase B (Akt) appears to play a key role in their neuroprotection. The present study aimed to investigate the effects of PROG (8,16 mg kg⁻¹) and ALLO (8,16 mg kg⁻¹) on the reversal of cognitive deficits induced by ketamine (30 mg kg⁻¹) via the PGRMC1 pathway in rat brains, including hippocampus and prefrontal cortex (PFC). Cognitive performance was evaluated by Morris water maze (MWM) test. Western blot and real-time quantitative polymerase chain reaction were utilized to assess the expression changes of protein and mRNA. Additionally, concentrations of PROG and ALLO in plasma, hippocampus and PFC were measured by a liquid chromatography-tandem mass spectrometry method. We demonstrated that PROG or ALLO could reverse the impaired spatial learning and memory abilities induced by ketamine, accompanied with the upregulation of PGRMC1/EGFR/GLP-1R/PI3K/Akt pathway. Additionally, the coadministration of AG205 abolished their neuroprotective effects and induced cognitive deficits similar with ketamine. More importantly, PROG concentrations were markedly elevated in PROG-treated groups in hippocampus, PFC and plasma, so as for ALLO concentrations in ALLO-treated groups. Interestingly, ALLO (16 mg kg⁻¹) significantly increased the levels of PROG. These findings suggest that PROG can exert its neuroprotective effects via activating the PGRMC1/EGFR/GLP-1R/PI3K/Akt pathway in the brain, whereas ALLO also restores cognitive deficits partially via increasing the level of PROG in the brain to activate the PGRMC1 pathway.
... Exogenous AlloP can improve experimental outcomes, including reductions to infarct volume, gliosis, cytokine upregulation, and improvements to post-recovery cognitive performance (Djebaili et al., 2005;Melcangi and Panzica, 2014;Sayeed et al., 2009). In vitro, AlloP is neuroprotective against hypoxic or excitotoxic insults (Frank and Sagratella, 2000). These effects are likely due in part to its pharmacodynamic profile as a potent positive allosteric modulator of GABA A receptors [and direct agonist in high concentration (Callachan et al., 1987)] and as an antagonist of Ltype Ca 2+ channels (Earl and Tietz, 2011;Hu et al., 2007), readily restoring tonic inhibition within neural networks. ...
Pregnane steroids, particularly allopregnanolone (AlloP), are neuroprotective in response to central insult. While unexplored in vivo, AlloP may confer protection against the neurological dysfunction associated with human immunodeficiency virus type 1 (HIV-1). The HIV-1 regulatory protein, trans-activator of transcription (Tat), is neurotoxic and its expression in mice increases anxiety-like behavior; an effect that can be ameliorated by progesterone, but not when 5α-reduction is blocked. Given that Tat's neurotoxic effects involve mitochondrial dysfunction and can be worsened with opioid exposure, we hypothesized that Tat and/or combined morphine would perturb steroidogenesis in mice, promoting neuronal death, and that exogenous AlloP would rescue these effects. Like other models of neural injury, conditionally inducing HIV-1 Tat in transgenic mice significantly increased the central synthesis of pregnenolone and progesterone's 5α-reduced metabolites, including AlloP, while decreasing central deoxycorticosterone (independent of changes in plasma). Morphine significantly increased brain and plasma concentrations of several steroids (including progesterone, deoxycorticosterone, corticosterone, and their metabolites) likely via activation of the hypothalamic-pituitary-adrenal stress axis. Tat, but not morphine, caused glucocorticoid resistance in primary splenocytes. In neurons, Tat depolarized mitochondrial membrane potential and increased cell death. Physiological concentrations of AlloP (0.1, 1, or 10 nM) reversed these effects. High-concentration AlloP (100 nM) was neurotoxic in combination with morphine. Tat induction in transgenic mice potentiated the psychomotor effects of acute morphine, while exogenous AlloP (1.0 mg/kg, but not 0.5 mg/kg) was ameliorative. Data demonstrate that steroidogenesis is altered by HIV-1 Tat or morphine and that physiological AlloP attenuates resulting neurotoxic and psychomotor effects.
... In the process of endogenous AlloP synthesis, cholesterol translocation to mitochondrial inner membrane by translocator protein 18 kD (TSPO) [121] and the catalytic reaction by 5α-reductase (5aRD) [122,123] are considered rate-limiting steps. AlloP potentiates the activity of GABA A receptors [124], and exerts neuroprotective properties both in in vitro cell culture [125][126][127][128] and in vivo animal models [129][130][131]. AlloP also increases myelination [132][133][134], enhances neurogenesis [135], decreases inflammation [134,136,137], and reduces apoptosis [138][139][140]. ...
Glaucoma is one of the most frequent causes of visual impairment worldwide and involves selective damage to retinal ganglion cells (RGCs) resulting in degeneration of neural pathways connecting retina to visual cortex. It is of interest that similarities in pathological changes have been described in Alzheimer's disease (AD), the most common cause of progressive memory loss and dementia in older people. Accumulation of amyloid-beta (Abeta) and hyperphosphorylated tau is thought to contribute to apoptotic neuronal death in Alzheimer's disease, and similar changes have been linked to apoptotic RGC death in glaucoma. Both glaucoma and Alzheimer's disease also suffer from a lack of effective treatments prompting a search for novel therapeutic interventions. Neurosteroids (NSs) (including oxysterols) are endogenous molecules synthesized in the nervous system from cholesterol that can modulate glutamate and GABA receptors, the primary mediators of fast excitatory and inhibitory neurotransmission in the brain, respectively. Because changes in the glutamate and GABA neurotransmitter systems contribute to the pathogenesis of AD and glaucoma, NSs are possible therapeutic targets for these disorders. In this review, we present recent evidence supporting pathological links between Alzheimer's disease and glaucoma, and focus on the possible role of NSs in these diseases and how NSs might be developed for therapeutic purposes.
... These derivatives are also able to modulate local neuronal activity. NMDA-induced excitotoxicity was diminished by allopregnanolone in several models including hippocampal neurons, rat embryonic cerebral cortical neurons and human NT2 neurons [51][52][53]. Furthermore, P19-N differentiated neurons treated with allopregnanolone were protected from NMDA-induced apoptotic cell death, preserving cytochrome c in the mitochondrion and Bax in the cytoplasm [54]. ...
Background:
Neurosteroids form the unique group because of their dual mechanism of action. Classically, they bind to specific intracellular and/or nuclear receptors, and next modify genes transcription. Another mode of action is linked with the rapid effects induced at the plasma membrane level within seconds or milliseconds. The key molecules in neurotransmission are calcium ions, thereby we focus on the recent advances in understanding of complex signaling crosstalk between action of neurosteroids and calcium-engaged events.
Methods:
Short-time effects of neurosteroids action have been reviewed for GABAA receptor complex, glycine receptor, NMDA receptor, AMPA receptor, G protein-coupled receptors and sigma-1 receptor, as well as for several membrane ion channels and plasma membrane enzymes, based on available published research.
Results:
The physiological relevance of neurosteroids results from the fact that they can be synthesized and accumulated in the central nervous system, independently from peripheral sources. Fast action of neurosteroids is a prerequisite for genomic effects and these early events can significantly modify intracellular downstream signaling pathways. Since they may exert either positive or negative effects on calcium homeostasis, their role in monitoring of spatio-temporal Ca2+ dynamics, and subsequently, Ca2+-dependent physiological processes or initiation of pathological events, is evident.
Conclusion:
Neurosteroids and calcium appear to be the integrated elements of signaling systems in neuronal cells under physiological and pathological conditions. A better understanding of cellular and molecular mechanisms of nongenomic, calcium-engaged neurosteroids action could open new ways for therapeutic interventions aimed to restore neuronal function in many neurological and psychiatric diseases.
... Furthermore, mutations of Glu73 alter the local membrane thickness 30 suggesting that this residue may play an important role in mediating putative effects of steroids and other lipids on VDAC structure and function. Neurosteroids have neuroprotective effects 32 , and neurosteroid modulation of VDAC may contribute to this action 25 . KK123 labeling of Glu73 suggests that neurosteroids bind to VDAC at a different site than cholesterol or propofol, which have been shown to photolabel and/or bind elsewhere in the protein 9,20 . ...
Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that depend on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs) where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a Fast Ligand Identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. FLI-tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. FLI-tag was coupled to an alkyne-containing neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of CID fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using FLI-tag and ETD we identified Glu73 as the site of photo-incorporation of our neurosteroid ligand in the IMP, mVDAC1, and top-down MS confirmed a single site of photolabeling.
... 13 Neuroprotection is a consequence of GABA A receptor--mediated hyperpolarization resulting in reduced neuronal excitation with subsequent reduced susceptibility to excitotoxic cell death. 33 Increases in neurosteroid concentration might also limit brain injury in the fetus. Increased allopregnanolone concentration within the fetal brain is thought to reduce the adverse effects of hypoxia, protecting against further insults and limiting cell death. ...
Neonatal encephalopathy is the most common neurologic condition affecting newborn foals and shares similarities with perinatal asphyxia syndrome of human infants. However, in many cases of neonatal encephalopathy there is no obvious episode of acute or chronic hypoxia and other mechanisms likely play a role in the pathogenesis. Increased concentrations of neuroactive progestagens are found in affected foals; whether these molecules are protective, as has been suggested, or play a role in the pathogenesis is unknown. Neurologic diseases other than neonatal encephalopathy affect foals occasionally and should be considered when evaluating sick foals with clinical signs of neurologic dysfunction.
... 26 Allopregnanolone is a neurosteroid that can be locally synthesized in the central nervous system (CNS) and a powerful modulator of neuronal excitability and neurotoxicity. 27 Allopregnanolone potentiates the activity of GABA A receptors, 28 and has neuroprotective properties in vitro [29][30][31][32] and in vivo. [33][34][35] Potential clinical targets for allopregnanolone 36,37 include mood and anxiety disorders, 38 alcoholism, 39 sleep disorders, 40 traumatic brain injury, 41,42 and neurodegenerative disorders. ...
Purpose:
Allopregnanolone is a neurosteroid and powerful modulator of neuronal excitability. The neuroprotective effects of allopregnanolone involve potentiation of γ-aminobutyric acid (GABA) inhibitory responses. Although glutamate excitotoxicity contributes to ganglion cell death in glaucoma, the role of GABA in glaucoma remains uncertain. The aim of this study was to determine whether allopregnanolone synthesis is induced by high pressure in the retina and whether allopregnanolone modulates pressure-mediated toxicity.
Methods:
Ex vivo rat retinas were exposed to hydrostatic pressure (10, 35, and 75 mm Hg) for 24 hours. Endogenous allopregnanolone production was determined by liquid chromatography and tandem mass spectrometry (LC-MS/MS) and immunochemistry. We also examined the effects of allopregnanolone, finasteride, and dutasteride (inhibitors of 5α-reductase), picrotoxin (a GABA(A) receptor antagonist), and D-2-amino-5-phosphonovalerate (APV, a broad-spectrum N-methyl-D-aspartate receptor [NMDAR] antagonist).
Results:
Pressure loading at 75 mm Hg significantly increased allopregnanolone levels as measured by LC-MS/MS. Elevated hydrostatic pressure also increased neurosteroid immunofluorescence, especially in the ganglion cell layer and inner nuclear layers. Staining was negligible at lower pressures. Enhanced allopregnanolone levels and immunostaining were substantially blocked by finasteride, but more effectively inhibited by dutasteride and APV. Administration of exogenous allopregnanolone suppressed pressure-induced axonal swelling in a concentration-dependent manner, while picrotoxin overcame these neuroprotective effects.
Conclusions:
These results indicate that the synthesis of allopregnanolone is enhanced mainly via NMDARs in the pressure-loaded retina, and that allopregnanolone diminishes pressure-mediated retinal degeneration via GABAA receptors. Allopregnanolone and other related neurosteroids may serve as potential novel therapeutic targets for the prevention of pressure-induced retinal damage in glaucoma.
... The question of whether low cholesterol contributes to faster PD progression, is merely a marker of more advanced pathology of PD, or is simply an epiphenomenon, cannot be addressed from this study. Cholesterol is involved in a plethora of critical biological functions ranging from cellular repair or degeneration171819202122 to being a neurosteroid precursor23242526. Whereas cholesterol may affect PD etiology or clinical progression, it is also conceivable that lower cholesterol could be a mere marker for a more advanced pathology of PD. ...
Recent studies have suggested that higher serum cholesterol may be associated with lower occurrence of Parkinson's disease (PD). This study is to test the hypothesis that higher serum cholesterol correlates with slower PD progression.
Baseline non-fasting serum total cholesterol was measured in 774 of the 800 subjects with early PD enrolled between 1987 and 1988 in the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) trial. Participants were followed for up to two years, with clinical disability requiring levodopa therapy as the primary endpoint. Hazard ratios (HRs) and 95% confidence intervals (CI) were determined for increasing serum cholesterol concentration (in quintiles) for clinical disability requiring levodopa therapy, after adjusting for confounders. At baseline, only nine subjects reported use of cholesterol-lowering agents (two with statins).
The overall mean cholesterol level was 216 mg/dL (range 100-355). The HR of progressing to the primary endpoint decreased with increasing serum cholesterol concentrations. Compared to the lowest quintile, the HRs (95%CI), for each higher quintile (in ascending order) are 0.83 (0.59-1.16); 0.86 (0.61-1.20); 0.84 (0.60-1.18); and 0.75 (0.52-1.09). The HR for one standard deviation (SD) increase = 0.90 [(0.80-1.01), p for trend = 0.09]. This trend was found in males (HR per SD = 0.88 [(0.77-1.00), p for trend = 0.05], but not in females [HR = 1.03 (0.81-1.32)].
This secondary analysis of the DATATOP trial provides preliminary evidence that higher total serum cholesterol concentrations may be associated with a modest slower clinical progression of PD, and this preliminary finding needs confirmation from larger prospective studies.
... An important consideration is whether variations in P 4 metabolism may underlie the protective effects of P 4 in different animal models of neural insult . In the central nervous system, the neuroprotective effects of P 4 (Frye, 1995a;Frank and Sagratella, 2000;Frye and Scalise, 2000;Lockhart et al., 2002;Ciriza et al., 2004;Djebaili et al., 2004;He et al., 2004;Rhodes and Frye, 2004;Rhodes, McCormick, and Frye, 2004;Djebaili et al., 2005;Rhodes and Frye, 2005a) are partly mediated by its metabolites subsequent to their rapid conversion by 5a-reductase and 3a-hydroxysteroid dehydrogenase to DHP and 3a,5a-THP (Mellon, Griffin, and Compagnone, 2001), respectively. Indeed, P 4 can have beneficial effects along a continuum of function (i.e., from improving memory to having neuroprotective effects against damage), and these effects are likely due to actions of its metabolites. ...
The nature of progesterone (P₄)'s neuroprotective effects is of interest. We investigated effects of P₄ when administered before, or after, kainic acid, which produces ictal activity and damage to the hippocampus, to mediate effects on spatial performance. The hypothesis was that P₄, compared with vehicle, would reduce decrements in Morris Water Maze performance induced by kainic acid. Experiment 1: We examined the effects of kainic acid on plasma stress hormone, corticosterone, and progestogen (P₄ and its metabolites) levels in plasma and the hippocampus after subcutaneous (s.c.) P₄ administration to ovariectomized rats. Rats administered kainic acid had the highest corticosterone levels immediately following injection. P₄ is 5α-reduced to dihydroprogesterone (DHP) and subsequently metabolized to 5α-pregnan-3α-ol-20-one (3α,5α-THP) by 3α-hydroxysteroid dehydrogenase. The regimen of P₄ used produced circulating and hippocampal levels of P₄, DHP, and 3α,5α-THP within a physiological range, which declined at 14 hours postinjection and were not altered by kainic acid. Experiment 2: The physiological P₄ regimen was administered to rats before, or after, kainic acid-induced seizures, and later effects on water maze performance were compared with that of rats administered vehicle. Rats administered kainic acid had significantly poorer performance in the water maze (i.e., increased latencies and distances to the hidden platform) than did rats administered vehicle. Administration of P₄ before, but not after, kainic acid prevented these performance deficits. Thus, these data suggest that a physiological regimen of P₄ can prevent some of the deficits in water maze performance produced by kainic acid.
... Based on the data reviewed here and elsewhere, 6,7,31,35,36,81,199,201,[232][233][234][235][236][237][238][239][240][241][242] we propose a model in which stress-induced HPA axis activation, or chronic exposure to elevated levels of GCs even in the absence of stress, sets in motion multiple pathways that ultimately endanger neurons or glial cells in the brain, may accelerate aging in certain cells in the brain and periphery and may contribute to various psychiatric symptoms (Fig. 2). First, elevated GC activity is associated with down-regulation of GRs, leading to some degree of GC resistance. ...
Elevated circulating levels of glucocorticoids are associated with psychiatric symptoms across several different conditions. It remains unknown if this hormonal abnormality is a cause or an effect of the psychiatric conditions. For example, the hypercortisolemia observed in a subset of patients with depression may have a direct impact on the symptoms of depression, but it is also possible that the hypercortisolemia merely reflects the stress associated with depression. Further, rather than causing depression, hypercortisolemia could represent a homeostatic attempt to overcome glucocorticoid resistance. Each of these possibilities will be considered, and correlational and causal evidence will be reviewed. This article will focus on the relationships between glucocorticoids and psychiatric symptoms in Cushing's syndrome, major depression, and steroid psychosis/steroid dementia, as well as the effects of exogenously administered glucocorticoids in normal volunteers. Similarities and differences in the relationship of glucocorticoid hormones to psychiatric symptoms in these conditions will be reviewed. Possible mediators of glucocorticoid effects on the brain and behavior, as well as possible "pro-aging" effects of glucocorticoids in certain cells of the body, will be reviewed. The article concludes with a conceptual model of glucocorticoid actions in the brain that may lead to novel therapeutic opportunities.
... The production rate and levels of neurosteroids decline rapidly with age, increasing the vulnerability of neurons to neurodegenerative processes [2]. Therefore, it seems that DHEA, its sulfate ester (DHEAs), and allopregnanolone (allo) are able to protect central nervous system (CNS) neurons against the neurotoxic action of N-methyl-d-aspartate (NMDA) [3,4] and to decrease cell death and cognitive defi cits after traumatic brain injury [5,6]. ...
To evaluate the effect of dehydroepiandrosterone (DHEA) as a neurosteroid on the rate of neurogenesis, neural survival, and proliferation of pluripotent stem cell-derived neurons, we have added DHEA to mouse P19 embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors (ECC- and ESC-NPs). In ECC-derived NPs, flow cytometric analysis of nestin and Tuj1-positive cells revealed that the percentages of these cells increased significantly for the markers following DHEA treatment of the cells. Moreover, the percentages of tyrosine hydroxylase (TH)-positive cells, the marker of dopaminergic neurons, significantly increased in the presence of DHEA. The expression of neural-specific genes such as Mash1, Pax6, Tuj1, and TH was also detected by RT-PCR analysis. BrdU incorporation and estrogen receptor (EsR) were found to be increased after DHEA induction. Moreover, apoptosis was significantly decreased after DHEA treatment. DHEA effect was also confirmed on human ESC-NPs by the enhancement of Tuj1- and TH-immunofluorescent-positive cells and TH and Nurr1 transcripts, as detected by quantitative RT-PCR. In conclusion, these results have presented evidence that DHEA was able to induce neurogenesis in mouse ECC and human ESC-NPs. This observation was related to the division of NPs and the reduction of apoptosis. Moreover, DHEA has dopaminergic potential in the cells of both orders. This provides a better insight into the differentiation and maintenance of neural cells and treatment of a wide variety of neurological diseases such as Alzheimer's and Parkinson's by stem cells.
... Progesterone administration at the dose used in the present study results in a significant increase in the brain levels of DHP and THP (Ciriza et al., 2006). The reduced metabolites of progesterone mediate several of the effects of the hormone in the brain, including affective, cognitive, and neuroprotective actions (Frye, 1995;Frank and Sagratella, 2000;Frye and Scalise, 2000;Ciriza et al., 2004Ciriza et al., , 2006Djebaili et al., 2004Djebaili et al., , 2005He et al., 2004;Rhodes and Frye, 2004;Rhodes et al., 2004). Our findings showing that the reduced derivatives of progesterone affect the phosphorylation of ERK-1/2 and Akt and the expression of PI3K in some brain regions suggest that progesterone metabolism may be involved in the hormonal effects on the MAPK and PI3K signaling pathways. ...
Several growth factors, such as vascular endothelial growth factor, brain-derived neurotrophic factor, and insulin-like growth factor-I are involved in the actions of progesterone in the central nervous system. Previous studies in neuronal and glial cultures have shown that progesterone may regulate growth factor signaling, increasing the phosphorylation of extracellular-signal regulated kinase (ERK) and the phosphorylation of Akt, components of the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3 kinase (PI3K) signaling pathways, respectively. In this study, we have evaluated whether progesterone and its reduced metabolites, dihydroprogesterone and tetrahydroprogesterone, regulate PI3K and MAPK signaling in the brain of ovariectomized rats in vivo. Significant increases in the phosphorylation of ERK, in the expression of the catalytic (p110) and the regulatory (p85) subunits of PI3K and in the phosphorylation of Akt were observed in the hypothalamus, the hippocampus, and the cerebellum 24 hr after progesterone administration. Progesterone metabolites partially mimicked the effect of progesterone and had a stronger effect on MAPK and PI3K signaling in the hypothalamus than in the other brain regions. These findings suggest that progesterone regulates MAPK and PI3K signaling pathways in the central nervous system in vivo by direct hormonal actions and by mechanisms involving progesterone metabolites.
... In humans, levels of allopregnanolone are low in depressed patients, and serotonin specific reuptake blocker (SSRI) antidepressant treatment increases CSF levels of this hormone, in direct proportion to the antidepressant effect (Uzunov et a1 1996; Uzunova et a1 1998 ). In addition to allopregnanolone , pregnenolone, THDOC and androsterone are promising neurosteroid targets for novel antidepressant or neuroprotective agent development (Barrot et a1 1999; Frank and Sagratella 2000; George et a1 1994; Maurice et a1 1999; Meieran et a1 In Review; Patchev et a1 1997; Rupprecht and Holsboer 1999a; Urbanoski et a1 2000). ...
Stress is commonly associated with a variety of psychiatric conditions, including major depression, and with chronic medical conditions, including diabetes and insulin resistance. Whether stress causes these conditions is uncertain, but plausible mechanisms exist by which such effects might occur. To the extent stress-induced hormonal alterations (e.g., chronically elevated cortisol levels and lowered dehydroepiandrosterone [DHEA] levels) contribute to psychiatric and medical disease states, manipulations that normalize these hormonal aberrations should prove therapeutic. In this review, we discuss mechanisms by which hormonal imbalance (discussed in the frameworks of "allostatic load" and "anabolic balance") might contribute to illness. We then review certain clinical manifestations of such hormonal imbalances and discuss pharmacological and behavioural treatment strategies aimed at normalizing hormonal output and lessening psychiatric and physical pathology.
... Neurosteroids, including AP, also have neuroprotective functions (8), particularly in the hippocampal region. AP administration exerts a potent neuroprotective effects in cell viability studies in rat hippocampus slices and in primary hippocampal cultures as the steroid attenuates glutamate-induced increases of intracellular calcium (46). Thus, high levels of AP may protect the fetal brain; for example, neurosteroids that protect neurons after hypoxic or asphyxic episodes could be beneficial to the developing fetus. ...
Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABAA receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5alpha-reductase type II (5alphaRII) enzymes in fetal sheep from 72 to 144 d gestation (term approximately 147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5alphaRII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5alpha-pregnan-3alpha-ol-20-one), a major sedative 5alpha-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (<50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth.
... There is now clear evidence that bioactive steroids, generally called neurosteroids, are synthesized within the nervous system of vertebrates (Baulieu et al. 1999;Mensah-Nyagan et al. 1999;Tsutsui et al. 2000;Mensah-Nyagan et al. 2001a,b;Mellon and Griffin 2002). Pharmacological and behavioural studies have suggested that neurosteroids are involved in the regulation of important neurobiological processes including cognition (Mayo et al. 1993;Vallée et al. 1997Vallée et al. , 2001, stress (Purdy et al. 1991;Patchev et al. 1996;Zimmerberg and Brown 1998), neuroprotection (Guth et al. 1994;Kimonides et al. 1998;Frank and Sagratella 2000), depression (Uzunova et al. 1998;Strö hle et al. 2000;Van Broekhoven and Verkes 2003) and anxiety (Majewska 1992;Brot et al. 1997;Hodge et al. 2002). However, the physiological significance of neurosteroidogenesis always remains poorly understood. ...
Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.
... Neurosteroids, including AP, have been shown to have neuroprotective functions (39), by modulation of the GABA A (40). GABA A receptor density has been shown to increase in the fetal brain between 85 and 128 d gestation such that adult concentrations are attained by 128 d. ...
Pregnane steroids have sedative and neuroprotective effects on the brain, due to interactions with the steroid-binding site of the GABAA receptor. In the adult brain, synthesis of the pregnane steroids is increased in response to stress. Therefore, we have used umbilicoplacental embolization to mimic chronic placental insufficiency during late gestation in sheep, to investigate the expression of the steroidogenic enzymes p450scc, 5alpha-reductase type I (5alphaRI), 5alpha-reductase type II (5alphaRII), and allopregnanolone (AP) content in the fetal brain. Umbilicoplacental embolization was induced from 114 d gestation (term approximately 147 d) by daily injection of inert microspheres into the umbilical artery and continued for 17-23 d. Fetal arterial oxygen saturation was reduced to approximately 60% of the preembolization value in each fetus, with a significant reduction in blood arterial Po2, pH, and plasma glucose concentrations (p < 0.05) and a significant increase in blood arterial Pco2 and plasma lactate concentrations (p < 0.05). At postmortem at 131-137 d gestation, embolized fetuses were growth-restricted (2.10 +/- 0.14 kg, n = 5) compared with age-matched controls (4.43 +/- 0.56 kg, n = 7, p < 0.05). Umbilicoplacental embolized fetuses showed increased P450scc expression in the primary motor cortex; 5alphaRI expression was not changed in any of the regions examined, whereas 5alphaRII expression was markedly increased in all brain regions. Brain AP content did not significantly change, whereas plasma concentrations were increased. These findings suggest that the increased expression of p450scc and 5alphaRII may be a response that maintains AP concentration in the fetal brain after compromised placental function and/or intrauterine stress.
... In addition to their actions as GABA A receptor modulators and anesthetics, neuroactive steroids are thought to have other biological activity. At the behavioral level, neuroactive steroids have neuroprotective effects (Claudio and Stefano, 2000). At the cellular level, the steroids have been5. ...
Neuroactive steroids modulate the function of gamma-aminobutyric acid type A (GABA(A)) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3alpha,5beta)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA(A) receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA(A) receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA(A) receptor function and anesthesia. Photolabeling of 35-kDa protein with [(3)H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA(A) receptor function as evidenced by radioligand ([(35)S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA(A) receptor function normally in VDAC-2-deficient fibroblasts transfected with alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA(A) receptor modulation or anesthesia.
... AP has potent neuroprotective functions (Majewska et al. 1986). It has been demonstrated to increase cell viability in studies with rat hippocampal slices and in primary hippocampal cultures as a result of the attenuation of glutamate-induced increases of intracellular Ca 2+ (Frank & Sagratella, 2000). This action arises J Physiol 560.2 through the modulation of the GABA A receptor leading to increased GABAergic inhibitory neurotransmission that reduces excitation and suppresses excitotoxicity induced by excessive glutamate stimulation. ...
Allopregnanolone (AP) is a potent modulator of the GABAA receptor. Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain. Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy. The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5alpha-reductase type II (5alphaRII) with brain and CSF neurosteroid concentrations. UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1-2 h. AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 10.4 +/- 0.4 and 12.4 +/- 0.3 to 26.0 +/- 5.1 and 27.6 +/- 6.4 nmol l(-1), respectively (P < 0.05), before returning to pre-occlusion levels by 3-4 h after UCO. When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid. AP, but not pregnenolone, was increased in CSF at this time. P450scc and 5alphaRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses. These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia. The action of the increased neurosteroid content at GABAA receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short-term protection to brain cells during such stress.
... The process of neurosteroid biosynthesis has been evidenced in the nervous system of several animal species and in humans, indicating that this process, which is well conserved through the vertebrate phylum, may control important neurophysiological functions (Mensah-Nyagan et al., 1999Tsutsui et al., 2000;Stoffel-Wagner, 2001;Schumacher et al., 2003). Pharmacological and behavioral studies have suggested that neurosteroids participate in the regulation of neurobiological mechanisms such as stress (Purdy et al., 1991;Patchev et al., 1996;Zimmerberg and Brown, 1998), anxiety (Majewska, 1992;Brot et al., 1997;Hodge et al., 2002), cognition (Mayo et al., 1993;Vallée et al., 1997Vallée et al., , 2001, and neuroprotection (Guth et al., 1994;Kimonides et al., 1998;Frank and Sagratella, 2000). In particular, several investigations using rodents as models revealed that within the nervous system, synthesis of progesterone (PROG) and its active metabolites, dihydroprogesterone (5-DHP) and tetrahydroprogesterone (3,5,THP), contributes to the protection of nerve cells against degeneration (Koenig et al., 1995;Ghoumari et al., 2003;Martini et al., 2003;Melcangi et al., 2003;Schumacher et al., 2004). ...
Studies performed with animals suggest neurosteroid involvement in neuroprotection. However in humans, the role of neurosteroidogenesis in the regulation of degenerative processes is unknown. To determine whether cellular factors intervening in degenerative mechanisms may interfere with the process of neurosteroidogenesis in humans, we combined pulse-chase experiments with HPLC and continuous flow scintillation detection to compare neurosteroid production in normal and transfected SH-SY5Y cells with key proteins involved in Alzheimer's disease (AD). Microscope analyses revealed that cell morphology was unchanged in stably transfected SH-SY5Y cells overexpressing human native tau (hTau40), mutant tau (P301L), and wild-type amyloid precursor protein (APPwt) compared to controls. Biochemical investigations showed that hTau40 enhanced progesterone (PROG), 17OHPROG, testosterone, and 3alpha-androstanediol neosynthesis from pregnenolone. In contrast, tau with the pathogenic P301L mutation was devoid of action on neurosteroidogenesis. Overexpression of APPwt inhibited PROG formation, did not affect 17OHPROG and testosterone, but increased 3alpha-androstanediol and estradiol synthesis. Extracellular treatment of control cells with aggregated amyloid peptide mimicked the action of APPwt expression on PROG but not on 3alpha-androstanediol and estradiol production. Moreover, PROG biosynthesis in APPwt cells was up-regulated in the presence of a gamma-secretase inhibitor. Our results provide the first evidence for the regulation of neurosteroid biosynthesis by key proteins involved in the etiology of AD. The data suggest that pathogenic factors may induce neurodegeneration in humans through the reduction of the synthesis of endogenous neuroprotective neurosteroids in nerve cells.
One of the most fascinating developments in the field of neuroscience in the second half of the 20th century was the discovery of the endogenous capacity of the brain for reorganization during adult life. Morphological and functional mechanisms underlying brain plasticity have been extensively explored and characterized. However, our understanding of the functional significance of these plastic changes is still fragmentary. This book shows that brain plasticity plays an essential role in the regulation of hormonal levels. The second aim is to propose that hormones orchestrate the multiple endogenous plastic events of the brain for the generation of adequate physiological and behavioral responses in adaptation to and in prediction of changing life conditions. The book starts by introducing the conceptual backgrounds on the interactions of hormones and brain plasticity. It then devotes itself to the analysis of the role of brain plasticity in the regulation of the activity of endocrine glands. It examines different hormonal influences on brain plasticity. Then, it goes on to cover the interactions of hormones and brain plasticity along the life cycle under physiological and pathological conditions.
Les hormones stéroïdes (dérivés du cholestérol) sont essentiellement synthétisées par les glandes endocrines (surrénales, gonades ou placenta). Cependant, le système nerveux, notamment neurones et cellules gliales possèdent la capacité de synthétiser de novo des « neurostéroïdes » à partir du cholestérol : c’est la neurostéroïdogenèse. Ces neurostéroïdes jouent un rôle essentiel sur le système nerveux en favorisant la neurogenèse, la prolifération des progéniteurs neuronaux, la régénération axonale, la myélinisation, la neuroprotection et la lutte contre l’apoptose neuronale. Ils ont également un rôle sur l’appétit, le comportement et les fonctions thymiques et cognitives. La prégnénolone et le sulfate de prégnénolone constituent des neurostéroïdes fondamentaux, non seulement en tant que précurseur de tous les composés stéroïdes ; mais également de par leurs actions propres nécessaires au bon fonctionnement du système nerveux. Le sulfate de prégnénolone est un neuromédiateur essentiel des systèmes de transmission synaptique GABA dépendant et glutamate dépendant, ainsi que du système de neurotransmission cholinergique avec la libération d’acétylcholine. Nous nous intéresserons dans cet article, au rôle central de la prégnénolone dans le système nerveux, et à ses implications dans les fonctions cognitives, le vieillissement cérébral et sa prévention, ainsi que la physiopathogénie de certaines affections neurologiques.
Allopregnanolone (A) and pregnanolone (P) are able to modify neural activities acting through the GABAA receptor complex. This capacity makes them useful as anticonvulsant, anxiolytic, or anti-stress compounds. In this study, the performance of seven synthetic steroids (SS) analogous of A or P containing an intramolecular oxygen bridge was evaluated using different assays. Competition assays showed that compounds 1, 5, 6 and 7 affected the binding of specific ligands for the GABAA receptor in a way similar to that of A and P, whereas compounds 3 and 4 stimulated [3H]-flunitrazepam and reduced [35S]-TBPS binding. The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) produces the precursor for A and P, and its activity is regulated by steroids. The action of several SS on 3β-HSD activity was tested in different tissues. All SS analyzed inhibit its activity, but compound 5 was the least effective. Finally, the neuroprotective role of two SS was evaluated in cerebral cortex and hippocampus cultures subjected to hypoxia. Glial fibrillary acidic protein (GFAP) increase was prevented by A, P, and compounds 3 and 5. Only A, P and compound 5 prevented neurofilament (NF160/200) decrease in hippocampus cultures, whereas A and compound 5 partially prevented NF200 and NF160 decreases respectively in cerebral cortex cultures. A prevented microtubule associated protein (MAP 2b) decrease in cerebral cortex cultures, while in hippocampus cultures only compounds 3 and 5 had effect. All steroids prevented MAP 2c decrease in both brain regions.
Neuroactive steroids such as allopregnanolone (3α-hydroxy-5α-pregnan-20-one) influence central nervous system (CNS) excitability by increasing GABA (gamma aminobutyric acid) inhibitory activity. Allopregnanolone concentrations are higher in the fetal compared to the adult ovine brain, suggesting that this neurosteroid may have a role in regulating fetal CNS activity during gestation. We examined the localisation of allopregnanolone-sensitive GABAA receptors in the fetal brain to determine if their sensitivity to allopregnanolone changed during late gestation. The binding of [35S] tert-butylbicyclophosothionate (TBPS) was used to identify the GABA-chloride ion receptor complex in fetal sheep brains at 90–95, 115–120 and 140–145 days gestational age (GA; term ~147 days), by autoradiography. Allopregnanolone (200 nM) reduced [35S]TBPS binding by 70–100% throughout the brain at all fetal ages examined. The levels of [35S]TBPS binding increased with advancing gestation in all regions examined except some areas of the medulla. Functionally related nuclei and brain areas associated with regulating somato/viscerosensory functions displayed high levels of [35S]TBPS binding from mid-gestation. These results indicate that allopregnanolone may interact with GABAA receptors to inhibit fetal CNS activity from mid-gestation. This inhibition may contribute to maintaining the sleep-like behaviour and low incidence of arousal-type activity typical of fetal life, and may be neuroprotective by limiting excitatory neurotransmission.
The term "neurosteroid" (NS) was introduced by Baulieu in 1981 to name a steroid hormone, dehydroepiandrosterone sulfate (DHEAS), that was found at high levels in the brain long after gonadectomy and adrenalectomy, and shown later to be synthesized by the brain. The term "neuroactive steroid" (NAS) refers to steroids that, independent of their origin, are capable of modifying neural activities. NASs bind and modulate different types of membrane receptors. The γ-amino butyric acid (GABA) and Sigma receptor complexes have been the most extensively studied, while glycine chloride channels, nicotinic acetylcholine receptors, and voltage-activated calcium channels, although less explored, are also modulated by NASs. Within the glutamate receptor family, N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainate receptors have been shown to be targets for NA and NAS modulation. Inside the neuron, Oxidized ring A reduced pregnanes, tetrahydroprogesterone (THP), and tetrahydrodeoxycorticosterone (THDOC) bind to the progesterone intracellular receptor (PR) and in this way can also regulate gene expression. Animal experimentation showed that cardinal symptoms of depression, anxiety, sleep disturbances, and memory dysfunctions are partly regulated by NAS. In turn, NAS and NS levels are modulated by psychotropic medications. NS levels, as well as NAS plasma concentrations, change in patients with depression syndromes, the levels return to normal baseline with recovery, but normalization is not necessary for successful therapy. Rather than with nosological syndromes and psychiatric disorders, NAS and NS steroid levels seem to correlate with specific symptoms. Hence, it will be profitable to search for and establish symptom-steroid relationships, as well as pharmacological and endogenous factors that can modulate NS biosynthesis and NAS formation, to be able to use these steroids in therapy.
Neurosteroids (NS) refers to steroids synthesized by the brain, neuroactive steroids (NAS) refers to steroids that, independent of their origin, are capable of modifying neural activities. NAS bind to and modulate different types of neurotransmitter receptors at the membrane level. The gamma-aminobutyric acid (GABA) and sigma receptor complexes have been the most extensively studied.
After intracellular oxidation, tetrahydroprogesterone (THP) to dihydroprogesterone (DHP), and tetrahydrodeoxycorticosterone (THDOC) to dihydrodeoxycorticosterone (DHDOC) bind to the progesterone intracellular receptor (PR), and in this way can also regulate membrane composition through gene expression.
Although NAS levels change in depression syndromes, in particular THP decreases while THDOC increases, levels return to normal baseline with recovery; however, normalization is not necessary for successful therapy.
Studies attempting to pinpoint the possible use of NAS levels as biological markers were unsuccessful. Data from studies on the evolution of nervous systems, evolutionary developmental biology, as well as anatomical and physiological findings, preclude that the disorders that result when the brain succumbs to psychiatric ailments can be categorically classified.
Persistence in maintaining essentialist classifications may help to explain why, until now, the search for biological markers in psychiatry has been an unrewarding effort. It is proposed that it would be more fruitful to focus on relationships between NAS and symptoms of psychiatric disorders, rather than with typologically defined disorders.
Neuronal cell death caused by pathophysiological over-activation of glutamate receptors and the subsequent CaII overloading, has been implicated in neurodegeneration after stroke, cerebral trauma and epileptic seizures. Recent findings suggest that certain progesterone metabolites (neurosteroids) such as allopregnanolone and dehydroepiandrosterone can protect neuronal cells from such insults. In the present study, murine P19 cells were induced to differentiate into postmitotic neurons expressing specific neuronal markers, including GABA(A) and NMDA receptors. Activation of NMDA receptors in P19-N neurons resulted in excitotoxic cell death, which involved suppression of the phosphorylation of the survival kinase PKB/Akt. Allopregnanolone and DHEA induced a rapid and prolonged phosphorylation of the Akt kinase and they were able to reverse the NMDA-induced suppression of the PI3-K/Akt pathway. The specificity of the neuroprotective effects of these neurosteroids was confirmed by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, as well as by the GABA(A) receptor antagonist, bicuculline. The neurotoxic effect of NMDA on P19-N neurons was directly correlated with increased CaII entry, since the addition of EGTA or BAPTA-AM, significantly suppressed the NMDA-induced decrease of phospho-Akt and subsequent neuronal death. These results suggest that neurosteroids are able to act as survival factors on P19-N neurons, promoting the activation of the PI3-K/Akt pathway through a calcium-entry dependent mechanism.
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.
It is well known that progesterone is synthesised and metabolised within the nervous system, and that one of its metabolites, allopregnanolone, potentiates the activity of GABA receptor anionic channels and modulates GABAergic neurotransmission. Progesterone is now under clinical trial for its neuroprotective properties, but its possible effects on neurotransmission have not yet been fully explored. The present study investigated acute effects of progesterone on the other major type of synaptic inhibition, glycinergic neurotransmission. Spontaneous glycinergic miniature currents were recorded in hypoglossal motoneurons, using the whole-cell patch-clamp technique in rat brainstem slices. A 20-min superfusion with progesterone (1 mum) triggered an increase in the frequency of glycinergic miniatures, whereas no effect of progesterone was observed after block with finasteride (5 mum) of 5alpha -reductase, the first enzymatic step leading from progesterone to allopregnanolone. The effect of progesterone could be mimicked by superfusion with allopregnanolone (0.3 mum), whereas no effect was induced by epiallopregnanolone. Thus, progesterone can increase the synaptic miniature release of glycine and this effect appears to be indirect, resulting from its metabolism into 5alpha-reduced derivatives, in particular into allopregnanolone. A low concentration of an exogenous GABA(A) agonist can also increase the frequency of inhibitory miniature currents in hypoglossal motoneurons. Thus, the effects of progesterone and allopregnanolone on glycine release can be at least partly explained by the potentiation of the activity of depolarizing presynaptic GABA receptor channels. The increase in the tonic synaptic release of a major inhibitory neurotransmitter should reduce the excitability of the neurons and contribute to their protection against excitotoxicity.
Unlabelled:
Perinatal asphyxia occurs in approximately 0.3% full-term newborn babies, and this percentage has not decreased despite medical advances. There are now evidences indicating that neurosteroids are important in neurodevelopment showing neuroprotective effects. We studied the potential protective effect of allopregnanolone (Allo) in vitro using organotypic cultures from neocortex, striatum, and hippocampus. Immunocytochemistry and confocal microscopy showed an increase of the glial fibrillary acidic protein (GFAP) signal in the studied brain areas after hypoxia. Western blot studies supported these results (hippocampus, 193%; neocortex, 306%; and striatum, 231%). Twenty-four-hour pretreatment with Allo showed different effects at the brain areas studied. In the hippocampus and the neocortex, 24-h pretreatment with Allo 5x10(-6) M showed to be neuroprotective as there was a significant decrease of the GFAP signal compared to control cultures exposed to hypoxia. Pretreatment with 5x10(-8) M Allo attenuated the astrogliosis response in the hippocampus and the neocortex in a nonsignificant way. Allo pretreatment at all doses did not show to affect the astrogliosis triggered by hypoxia in the striatum. Cell survival was analyzed by measuring LDH. After 1 h of hypoxia, all cultures showed a nonsignificant increase of LDH, which was greater after 24 h of hypoxia (hippocampus, 180%; striatum-cortex co-cultures, 140%). LDH levels have no changes by Allo pretreatment before hypoxia.
Conclusion:
24 h pretreatment with 5x10(-6) M of Allo does not change neuronal viability but it prevents astrogliosis induced by hypoxia in the hippocampus and the neocortex.
In our minireview we summarize the neuroprotective effect of atypical antipsychotic and selective serotonin re-uptake inhibitors after cerebral ischemia. In regard of increasing rate of poststroke mood disorders and current evidences indicating to an increased rate of cerebrovascular accidents after neuroleptic usage by the elderly population we also reviewed the clinical relevance of the neuroprotective and mood stabilizing effect of atypical antipsychotic agents in the light of basic pathophysiology of stroke.
We have previously shown the neuroprotective effect of atypical antipsychotic agents by experimental cerebral ischemia. However the impact of their high dose related side effects on their low dosage related neuroprotectivity is still unknown.We evaluated the possible neuroprotective effects of high dose olanzapine (10mg/kg) treatment on ischemic brain injury 24 hr after permanent cerebral ischemia. Olanzapine showed neither a neuroprotective nor a neurotoxic effect after focal cerebral ischemia. This finding could suggest that dose related side effect of olanzapine could involve a restriction of its neuroprotective effect unlike lower doses that have been reported to have neuroprotective effect.
Some neurosteroids show neuroprotective action in in vitro and in vivo studies, but their interaction with apoptotic/necrotic processes has been only partially unraveled. The aim of the present study was to examine the effect of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), pregnenolone (PGL) and allopregnanolone (Allo) on staurosporine-, glutamate-, and NMDA-induced damage in primary cortical neuronal culture. DHEA, DHEAS and PGL (0.1 and 1 microM) inhibited the staurosporine-evoked LDH release and decreased the number of apoptotic cells as shown by Hoechst;s staining, whereas Allo was without effect. The neurosteroids affected neither the staurosporine-evoked changes in caspase-3 activity nor the decrease in mitochondrial membrane potential. It was also shown that protective effects of DHEA, DHEAS and PGL against staurosporine-induced LDH release were attenuated by extracellular signal-regulated kinase (ERK)--mitogen-activated protein kinase (MAPK) inhibitor--PD 98059 (5 microM) but not by phosphatidylinositol-3-kinase (PI3-K) inhibitors such as LY 294002 (1 microM) or wortmannin (10 nM). The involvement of ERK2-MAPK in protective effects of neurosteroids was confirmed by Western blot study. Further study demonstrated that glutamate-induced cell damage was attenuated by DHEA, DHEAS, and PGL, but not by Allo. None of the steroids influenced NMDA-induced LDH release. The results of the present in vitro studies suggest that excitatory neurosteroids DHEA, DHEAS and PGL at physiological concentrations participate in the inhibition of cortical neuronal degeneration elicited by staurosporine and glutamate, whereas the most potent positive modulator of GABA(A) receptor--Allo--has no effect. Moreover, neurosteroids appear to attenuate the staurosporine-induced cell damage in a caspase-3 independent way and their neuroprotective mechanism of action involves the increase in ERK-MAPK phosphorylation.
The brain, like the adrenals, gonads and the placenta, is a steroidogenic tissue. However, unlike classic steroidogenic tissues, the synthesis of steroids in the nervous system requires coordinated expression and regulation of genes encoding the steroidogenic enzymes in several different cell types (neurons and glia) at different locations in the nervous system, often at some distance from the cell bodies. Furthermore, the synthesis of these steroids might be developmentally regulated and related to their functions in the developing brain. The steroids synthesized by the brain and nervous system, given the name 'neurosteroids', have a wide variety of diverse functions. In general, they mediate their actions not through classic steroid hormone nuclear receptors, but through other mechanisms, such as ion-gated neurotransmitter receptors or direct/indirect modulation of other neurotransmitter receptors. We summarize the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their pharmacological properties and modes of action. The physiological relevance and potential uses of neurosteroids in certain human diseases are discussed.
Progesterone modulates gamma-aminobutyric acid and excitatory amino acid neurotransmitter systems and has neuroprotective properties in models of hypoxia-ischemia. This study examined the in vitro effects of allopregnanolone, the active progesterone metabolite, in models of N-methyl-D-aspartate (NMDA)-induced necrosis and apoptosis. Cultured NT2 neurons were exposed to 1 mM NMDA. Lactate dehydrogenase (LDH) release was measured 24 h later. NMDA at a concentration of 1 mM produced a 39 +/- 19% release of total LDH. Exposure to 10 microM allopregnanolone prior to NMDA exposure reduced LDH release by 51% (P = 0.0028). NMDA stimulated apoptotic cell changes defined by terminal dUTP nick-end labeling (TUNEL) and 5,5', 6,6'-tetrachloro-1,1,3,3'-tetra ethlybenzimidazolycarbocyanide iodide staining were reduced to baseline values by both 10 microM allopregnanolone and 100 microM MK-801. Pretreatment with allopregnanolone (0-10 microM) reduced the percentage of TUNEL-positive cells in a dose-dependent manner (EC(50) = 2.7 +/- 0.1 nM). Physiologic concentrations of allopregnanolone provided protection against both necrotic and apoptotic injury induced by NMDA excitotoxicity.
The neurosteroids allopregnanolone (5alpha-pregnan-3alpha-ol-20-one; 5alpha,3alpha-P) and its 5beta-epimer pregnanolone (5beta,3alpha-P), and pregnenolone sulfate (PS) were examined for effects on spontaneous epileptiform discharges induced by 100 microM picrotoxin (PTX) and 55 microM 4-aminopyridine (4-AP) in the CA3 region of the rat hippocampal slice. At a concentration of 10 microM, 5alpha,3alpha-P partially reduced PTX-induced bursting and at 30 and 90 microM completely suppressed bursting. In contrast, 100 microM 5beta,3alpha-P failed to alter the discharge frequency. 5alpha,3alpha-P depressed 4-AP-induced bursting with similar potency as in the PTX model; 100 microM 5beta,3alpha-P was also partially effective. In the 4-AP model, 5alpha,3alpha-P inhibited both the more frequent predominantly positive-going potentials as well as the less frequent negative-going potentials that may be generated by synchronous GABAergic interneuron firing. PS enhanced the PTX bursting frequency and, in the 4-AP model, increased the frequency of negative potentials but did not alter the frequency of positive potentials. By itself, PS did not induce bursting. The effects of the steroids in the in vitro seizure models largely correspond with their activities on GABA(A) receptors; suppression of discharges may occur as a result of direct activation of these receptors rather than modulation of GABA-mediated synaptic responses. PTX and 4-AP-induced bursting in the hippocampal slice are useful models for directly assessing neurosteroid effects on seizure susceptibility under conditions that eliminate the factor of brain bioavailability.
Aggressive behavior can serve important adaptive functions in social species. However, if it exceeds the species-typical pattern, it may become maladaptive. Very high or escalated levels of aggressive behavior can be induced in laboratory rodents by pharmacological (alcohol-heightened aggression), environmental (social instigation), or behavioral (frustration-induced aggression) means. These various forms of escalated aggressive behavior may be useful in further elucidating the neurochemical control over aggression and violence. One neurochemical system most consistently linked with escalated aggression is the GABAergic system, in conjunction with other amines and peptides. Although direct stimulation of GABA receptors generally suppresses aggression, a number of studies have found that positive allosteric modulators of GABAA receptors can cause increases in aggressive behavior. For example, alcohol, benzodiazepines, and many neurosteroids are all positive modulators of the GABAA receptor and all can cause increased levels of aggressive behavior. These effects are dose-dependent and higher doses of these compounds generally shift from heightening aggressive behavior to being sedative and anti-aggressive. In addition, these modulators interact with each other and can have additive effects on the GABAA receptor and on behavior, including aggression. The GABAA receptor is a heteropentameric protein that can be constituted from various subunits. It has been shown that subunit composition can affect sensitivity of the receptor to some modulators and that subunit composition differentially affects the sedative vs anxiolytic actions of benzodiazepines. Initial studies targeting alpha subunits of the GABAA receptor point to their significant role in the aggression-heightening effects of alcohol, benzodiazepines, and neurosteroids.
The ovarian hormone progesterone is neuroprotective in some animal models of neurodegeneration. Progesterone actions in the brain may partly be mediated by the locally produced metabolites 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone. The neuroprotective effects of these two metabolites of progesterone were assessed in this study. Ovariectomized Wistar rats were injected with kainic acid, to induce excitotoxic neuronal death in the hippocampus, and with different doses of 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone. The number of surviving neurones in the hilus of the dentate gyrus of the hippocampus was assessed with the optical dlisector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurones and in the induction of vimentin expression in reactive astrocytes, a sign of neural damage. Low doses of 5α-dihydroprogesterone (0.25 and 0.5 mg/kg body weight, b.w.) prevented the loss of hilar neurones and the appearance of vimentin immunoreactivity in astrocytes. Higher doses (1-2 mg/kg b.w.) were not neuroprotective. By contrast, low doses of 3α,5α-tetrahydroprogesterone (0.25-1 mg/kg b.w.) were unable to protect the hilus from kainic acid while higher doses (2-4 mg/kg b.w.) were protective. The different optimal neuroprotective doses of 5α-dihydroprogesterone and 3α, 5α-tetrahydroprogesterone suggest that these two steroids may protect neurones using different mechanisms. The neuroprotective effects of 3α, 5α-tetrahydroprogesterone may be exerted by the inhibition of neuronal activity via the GABAA receptor. This latter possibility is supported by the observation that 3β,5α-tetrahydroprogesterone, an isomer of 3α,5α-tetrahydroprogesterone that does not bind to GABAA receptor, was not neuroprotective. In summary, our findings suggest that progesterone neuroprotective effects may be, at least in part, mediated by its reduced metabolites 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone.
Progestins have neuroprotective effects in several in vitro models of neurodegeneration and in vivo in seizure models. The extent to which progesterone's in vivo protective effects may generalize to models not involving seizure processes and whether progesterone's protective effects are modulated by its metabolites have not been comprehensively investigated. The present experiments investigated the effects of progesterone and its metabolites, dihydryoprogesterone (DHP) and 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP), to protect the hippocampus from damage induced by adrenalectomy (ADX). In Experiments 1 and 2, progesterone, DHP, or 3alpha,5alpha-THP administration (1 mg/kg sc) to female (Experiment 1) or male (Experiment 2) rats similarly reduced the total number of ADX-induced pyknotic cells in the dentate gyrus compared with vehicle administration. In Experiment 3, blocking progesterone's metabolism to 3alpha,5alpha-THP with coadministration of a 5alpha-reductase inhibitor, finasteride (10 mg/kg sc), in female rats attenuated progesterone's protective effects on cell death in the dentate gyrus. Together, these data suggest that progestins can protect against ADX-induced cell death and that the actions of the progesterone metabolite, 3alpha,5alpha-THP, may underlie these effects.
In nondiabetic animals, estrogen has been shown to provide significant neuroprotection in focal and transient forebrain ischemia models. However, that neuroprotection may be diminished or lost in the diabetic. In this study, we compared the level of brain damage in intact, ovariectomized (OVX) and 17beta-estradiol (E(2))-treated OVX female rats rendered diabetic and chronically ( approximately 4 weeks) hyperglycemic via streptozotocin (STZ). Rats were subjected to 20 min of unilateral transient forebrain ischemia (reduction in cortical CBF to 20% of baseline). Neurologic function was analyzed daily and brain histopathology (in H&E-stained sections) was evaluated at 72 h of reperfusion. Supplemental histopathologic information was obtained from additional TUNEL-stained sections. When comparing neurologic outcome scores in the three groups, E(2)-treated OVX females displayed the highest degree of dysfunction and intact females the least (OVX rats not treated with E(2) were intermediate), with the difference between the intact and E(2)-treated groups being statistically significant. That same order was often observed with the regional histopathologic analyses of H&E-stained tissue. A significantly higher magnitude of neuronal loss in both OVX groups, when compared to intact females, was observed in the CA4 sector of the hippocampus and in the cortex. In addition, cell loss in the dorsal thalamus of the E(2)-treated group was significantly greater than in the intact females. Those results were generally corroborated by TUNEL-analysis, with 67% of the E(2)-treated, 33% of the control OVX, and only 17% of the intact females displaying TUNEL-positive cells in multiple regions. In conclusion, the present findings strongly suggest that the neuroprotective benefits of estrogen replacement therapy may be lost in the diabetic female rat.
Many functions have been attributed to neurosteroids including actions as anxiolytics, roles in myelination, inhibitors of neuronal toxicity and ischemia, and roles in neuronal growth and differentiation. To understand the functions of neurosteroids during nervous system development, we used two mouse models: one, in which the cyp17 gene was ablated, thus ablating synthesis of the neurosteroid DHEA, and a second, in a mouse model of a human childhood fatal neurodegenerative disease, Niemann-Pick Type C (NP-C). Cyp17-/- mice died unexpectedly approximately embryonic day 7. Cyp17 was expressed in the embryonic endoderm at E7, where 17alpha hydroxylase and c17,20 lyase activities were found. Hormonal replacement was ineffective in rescuing the embryos. The function of P450c17 and/or its steroid products in early mouse development is unknown. In the second model, we used a naturally-occurring NP-C mutant mouse. Mutations in the npc1 gene results in lysosomal accumulation of cholesterol and gangliosides in humans and in the mouse, which also recapitulates the onset of neurological deficits, neuronal loss and death typical of the most severe form of the human disease. We showed that there is a substantial reduction in the synthesis of the neurosteroid allopregnanolone (ALLO) at birth, which may lead to abnormal neural development. ALLO treatment was highly effective; ALLO-treated NP-C mice had substantially increased survival and delays in neurologic impairments, coinciding with marked improvements in neuronal survival, and reduction of gangliosides. These data suggest that neurosteroids play an important role in brain development and maturation and may be an effective therapy for NP-C and perhaps other lysosomal storage diseases.
The term "neurosteroid" (NS) was introduced by Baulieu in 1981 to name a steroid hormone, dehydroepiandrosterone sulfate (DHEAS), that was found at high levels in the brain long after gonadectomy and adrenalectomy, and shown later to be synthetized by the brain. Later, androstenedione, pregnenolone and their sulfates and lipid derivatives as well as tetrahydrometabolites of progesterone (P) and deoxycorticosterone (DOC) were identified as neurosteroids. The term "neuroactive steroid" (NAS) refers to steroids which, independent of their origin, are capable of modifying neural activities. NASs bind and modulate different types of membrane receptors. The GABA and sigma receptor complexes have been the most extensively studied, while glycine-activated chloride channels, nicotinic acetylcholine receptors, voltage-activated calcium channels, although less explored, are also modulated by NASs. Within the glutamate receptor family, N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainate receptors have also been demonstrated to be a target for steroid modulation. Besides their membrane effects, once inside the neuron oxidation of Ring A reduced pregnanes, THP and THDOC, bind to the progesterone intracellular receptor and regulate gene expression through this path. The involvement of NASs on depression syndromes, anxiety disorders, stress responses to different stress stimuli, memory processes and related phenomena such as long-term potentiation are reviewed and critically evaluated. The importance of context for the interpretation of behavioral effects of hormones as well as for hormonal levels in body fluids is emphasized. Some suggestions for further research are given.
A plasma biomarker for neurodegenerative disease is desirable because blood is relatively simple to obtain compared with other biological samples such as cerebrospinal fluid. Recent literature suggests that neurosteroid metabolism may be altered in Alzheimer's disease (AD).
We sought to measure the plasma levels of seven steroids to assess their potential as biomarkers for dementia and AD. Methods: Steroids were measured using validated radioimmunoassay methods in AD (n=15), non-AD dementia (n=4), and control subjects (n=20). Demented subjects were in the mild-to-moderate stages of illness. Measurements were done blind to subject status in an independent laboratory.
The notable finding was the significantly lower 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) level in demented subjects compared with controls (25% decrease; p=0.004); 3alpha,5alpha-THP was the only one of the steroids demonstrating an effect of dementia.
Lowered 3alpha,5alpha-THP levels appear promising as a biomarker in dementia, but further work is needed to establish the sensitivity and specificity of these findings in AD.
Progesterone and its metabolites are potent allosteric modulators of GABA(A) receptor function, through a direct, non-genomic interaction with specific receptor subtypes. In addition, fluctuations in the concentration of progesterone, and allopregnanolone in particular, have been shown to modulate GABA(A) receptor gene expression and activity. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons which express specific neuronal markers, including GABA(A) and N-methyl-d-aspartate (NMDA) receptors. Apoptotic cell death, induced in the presence of NMDA, was efficiently prevented by allopregnanolone and dehydroepiandrosterone (DHEA) but not DHEA sulfate. Apoptosis was accompanied by cytochrome c release to the cytoplasm and Bax translocation to the mitochondria, while the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL remained unchanged. In the presence of the most potent neurosteroid, allopregnanolone, DNA fragmentation as well as cytochrome c and Bax translocation were prevented. On the other hand, short-term exposure (1-20 microm, 24 h) of P19-derived neurons to allopregnanolone and DHEA significantly increased the levels of alpha1 and beta2 mRNAs of GABA(A) receptor, while the levels of NR1 mRNA of NMDA receptor were not altered. These results suggest that neurosteroids, interfering with the mitochondrial apoptotic pathway, are able to act as survival factors in neuronal cells, while they contribute to GABA(A) receptor plasticity modulating the expression of its subunits.
The spinal cord (SC) is a biosynthetic center for neurosteroids, including pregnenolone (PREG), progesterone (PROG), and 3alpha/5alpha-tetrahydroprogesterone (3alpha/5alpha-THP). In particular, an active 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 P450sccpositive 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]CHOLwas 80% higher in the DH of neuropathic rats. Radioimmunoassays showed an increase of PREG and 3alpha/5alpha-THP concentrations in neuropathic rat DH. The upregulation of PREG and 3alpha/5alpha-THP biosynthesis might be involved in endogenous mechanisms triggered by neuropathic rats to cope with the chronic pain state. 3alpha/5alpha-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.
To analyze the functional consequences of hypoxia on the efficacy of intracortical inhibitory mechanisms mediated by gamma-aminobutyric acid (GABA), extra- and intracellular recordings were obtained from rat primary somatosensory cortex in vitro. Hypoxia, induced by transient N2 aeration, caused a decrease in stimulus-evoked inhibitory postsynaptic potentials (IPSPs), followed by a pronounced anoxic depolarization. Upon reoxygenation, the fast (f-) and long-latency (l-) IPSP showed a positive shift in the reversal potential by 24.4 and 14.9 mV, respectively. The peak conductance of the f- and l-IPSP was reversibly reduced in the postanoxic period by 72% and 94%, respectively. Extracellular field potential recordings and application of a paired-pulse inhibition protocol confirmed the enhanced sensitivity of inhibitory synaptic transmission for transient oxygen deprivation. Intracellular recordings from morphologically or electrophysiologically identified interneurons did not reveal any enhanced susceptibility for hypoxia as compared to pyramidal cells, suggesting that inhibitory neurons are not selectively impaired in their functional properties. Intracellularly recorded spontaneous IPSPs were transiently augmented in the postanoxic period, indicating that presynaptic GABA release was not suppressed. Developmental studies in adult (older than postnatal day 28), juvenile (P14-18), and young (P5-8) neocortical slices revealed a prominent functional resistance of immature tissue for hypoxia. In comparison with adult cortex, the hypoxia-induced reduction in excitatory and inhibitory synaptic transmission was significantly smaller in immature cortex. Our data indicate a hypoxia-induced distinct reduction of postsynaptic GABAergic mechanisms, leading to the manifestation of intracortical hyperexcitability as a possible functional consequence.
1.1. Superfusion of cultured hippocampal cells with glutamate (Glu) 0.5 mM for 5 min induces an increase of [Ca2+i] that is quickly followed by a recovery to control level. Addition of dynorphin or D-pen2-D-pen5-enkephalin (DPDPE) induces a persistence of the elevated [Ca2+i], while [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) does not influence it.2.2. Superfusion with Glu for 10 min induces a persistent increase of [Ca2+i], that is partially reverted by DAMGO, but not affected by dynorphin or DPDPE.3.3. The author suggests a differential influence of selective opioids on the Glu-induced [Ca2+i] increase.
Anoxia induced by exposure to N(2) gas for 15, 30, 50 and 60 s showed appearance of varying degrees of restlessness, tremor and convulsive behaviour resulting in mortality of adult rats. Diazepam treatment in pre- and post-anoxic conditions (10 and 20 mg/kg, respectively) has been found to decrease the anoxia-induced mortality rate along with suppression of other abnormal behaviours. In anoxic rats an increase in glutamate (Glu) level was noted. There was no change in the activity of glutamic acid decarboxylase (GAD), but GABA-aminotransferase (GABA-T) activity decreased significantly. In diazepam-treated (pre- and post-anoxic) rats there was an increase in the GAD activity with concomitant increase in ?-aminobutyric acid (GABA) level. GABA-T activity as well as Glu level became normalized. Results indicate that diazepam treatment may have some protecting ability from anoxia-induced imbalance in rat brain glutamatergic and GABAergic functions.
This chapter discusses epilepsy and γ-aminobutyric acid (GABA) mediated inhibition. The inhibitory action of short-chain ω-amino acids (such as β-alanine, GABA, and taurine) on central neurons has been established by the microiontophoretic studies. Additional data that have led to the widespread acceptance of the hypothesis that GABA is the inhibitory transmitter at numerous central sites include (1) the demonstration of high GABA content and glutamic acid decarboxylase (GAD) activity in synaptosome preparations, (2) the evidence for the selective local release of GABA when known inhibitory systems are activated in the cerebral cortex or cerebellum, and (3) evidence from intracellular recordings that the changes in membrane potential and in specific ionic conductances produced by physiological inhibitory inputs are similar to those produced by the iontophoretic application of GABA. The principal sites where GABA produces hyperpolarization of the neuronal membrane, which resembles the physiologically induced inhibitory postsynaptic potential (IPSP) in its dependence on an enhanced chloride conductance, and where naturally induced inhibition and the action of applied GABA are blocked by picrotoxin or bicuculline, are the neocortex, the cerebellum, the hippocampus, and the thalamus.
The abundant CNS cholesterol and its sulfate derivative serve as precursors of different neurosteroids, which bidirectionally modulate neuronal excitability, by potentiating or inhibiting function of the GABAA receptors. The regulation of GABAA receptors in the CNS by the steroids of central or peripheral origin may constitute a vital means of brain-body communication, essential for integrated whole organism responses to external stimuli or internal signals. Modulation of the brain GABA receptors by neurosteroids may form the basis of a myriad of psychophysiological phenomena, such as memory, stress, anxiety, sleep, depression, seizures and others. Therefore, the aberrant synthesis of centrally-active steroids may contribute to defects in neurotransmission, resulting in a variety of neural and affective disorders. The biosynthesis of neurosteroids may also be altered by diet and certain psychotropic drugs, thereby affecting excitation of neurons. Hereditary differences in the level of synthesis and catabolism of different neurosteroids may underlie individual variations in CNS excitability, contributing to differences in personality traits, including the inherited susceptibility to drug addition.
Bilateral transient occlusion of carotid arteries in gerbils for 7 min results in delayed neuronal cell death in hippocampal field CA1. Local gamma-aminobutyric acid (GABA)ergic neurons survive the ischemic insult. Here we show that interneurons in gerbil hippocampus are parvalbumin-immunoreactive, that they contain the GABA-synthetizing enzyme glutamic acid decarboxylase (GAD), and that they are resistant to the effects of ischemia, being present up to 28 days after the insult. It might be concluded that the presence of the Ca2+-binding protein parvalbumin protects the GABAergic neurons from the deleterious consequences of ischemia-induced excitotoxin-mediated Ca2+-influx.
A pulse-paired stimulation technique was used to examine the in vitro effects of the isomers of etomidate on synaptic transmission between Schaffer collaterals and CA1 pyramidal cells in the guinea pig hippocampus. Etomidate produced a dose-related, stereospecific, reversible increase in paired-pulse inhibition. Replacement of Cl- by isethionate reversed the inhibition induced by (+)-etomidate. Together with earlier biochemical evidence, these results show that in the mammalian CNS (+)-etomidate enhances central inhibition by increasing the effectiveness of gamma-aminobutyric acid (GABA) in a chloride-dependent fashion.
In the CA1 subfield of the gerbil hippocampus, an unusual series of changes were noticed after ischemia. Mongolian gerbils were subjected to bilateral carotid occlusion for 5 min. Perfusion fixation was performed 3, 6 and 12 h or 1, 2, 4, 7 and 21 days afterwards. Specimens obtained from the dorsal hippocampus were processed for light and electron microscopy. Three different types of changes were observed in the CA4, CA2 and CA1 subfields. In CA4, the change was rapid and corresponded to ischemic cell change. The alteration in CA2 was relatively slow, and identical to what has been called reactive change. On the contrary, the change in the CA1 pyramidal cells was very slow, only becoming apparent by light microscopy 2 days following ischemia. The CA1 subfield was selected for electron microscopic observation. The lamellar alignment of proliferated cisterns of the endoplasmic reticulum was the most conspicuous finding in these cells. Four days following ischemia, almost all of the pyramidal cells in CA1 were destroyed. In the CA1 neuropil, numerous presynaptic terminals remained without being apposed to normal postsynaptic sites. These changes in CA1, called here 'delayed neuronal death', may differ from those thought to be typical of ischemic neuronal damage. It was unlikely that the disturbance of local blood vessels was the cause of these changes.
In the gasping test in mice, the hypobaric hypoxia test in mice, the hypoxic hypoxia test in rats and the histotoxic anoxia test in rats, etomidate gave a more intense protection than thiopental and methohexital. Contrary to the barbiturates, etomidate protects against hypoxia in subanesthetic doses.
Effects of enkephalins on hippocampal pyramidal cell activity were studied in situ and in the in vitro hippocampal slice. Active enkephalin derivatives produced a dose-dependent naloxone-reversible excitation in both preparations whereas inactive enkephalin derivatives had no effect. Several different types of experiments, carried out in the slice, strongly suggest that this excitation is due to blockade of inhibitory pathways. First, when the pyramidal cell population spike is increased during enkephalin administration, no change is seen in the simultaneously recorded EPSP. Second, the magnitude of the enkephalin effect is highly correlated with the amount of inhibition, as judged by paired-pulse stimulation, initially present in the slice. Third, if inhibitory pathways are depressed by a brief period of hypoxia, enkephalin has little effect. Finally, enkephalin responses are mimicked by picrotoxin, which selectively antagonizes inhibitory input to the pyramidal neuron. Since enkephalins do not block the effects of GABA, the putative inhibitory transmitter, these data suggest that opioid peptides depress the inhibitory interneurons and disinhibit the pyramidal cells.
Previously, research elucidating steroid hormone actions in the central nervous system has focused on their role in sexual reproduction and maintaining homeostasis. The hippocampus is a target of steroid modulation and is involved in the development of emotional behavior and memory storage. Area CA1 of the hippocampus contains a high density of androgen receptor (AR) and N-methyl-D-aspartate (NMDA) receptors. NMDA receptors underlie excitatory synaptic transmission and excitotoxicity in CA1 neurons. The effects of AR activation on the neurophysiology of hippocampal pyramidal neurons is unknown. Standard intracellular recording techniques in hippocampal slices were used to investigate the effects of the non-aromatizable androgen, 5-alpha-dihydrotestos-terone-proprionate (DHTP), on CA1 pyramidal cell characteristics and NMDA receptor-mediated responses. Male Sprague-Dawley rats were unoperated, sham-operated (SHAM), gonadectomized (GDX), or gonadectomized with DHTP replacement therapy (GDX + DHTP). Neuronal AR was saturated by DHTP treatment as determined by binding studies and immunocytochemistry. Chronic DHTP treatment increased the action potential duration and decreased the fast afterhyperpolarization (fAHP) amplitude. To test the effect of DHTP on glutamate receptor-mediated responses, hippocampal slices were exposed to increasing concentrations of NMDA. In pyramidal cells from SHAM and GDX-treated animals, 30 microM NMDA induced an irreversible depolarization; the membrane potential of pyramidal cells from GDX + DHTP-treated animals recovered to baseline. The effect of DHTP was time dependent, implicating protein synthetic mechanisms. Our findings demonstrate that androgens can influence pyramidal cell characteristics and neurotransmitter-evoked actions in hippocampal CA1 pyramidal cells.
Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3alpha-ol-5beta-pregnan-20-one hemisuccinate (3alpha5betaHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3alpha5betaHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3alpha5betaHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3alpha5betaHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.
In the present study we examined effects of some neurosteroids on the kainate-induced seizures, lethality and neurotoxicity in mice. We found that 5 alpha-pregnan-3 alpha-ol-20-one (allopregnanolone; 10 and 20 mg/kg) markedly elevated CD50 for kainate-induced convulsions, whereas 5 beta-pregnan-3 alpha-ol-20-one, 5 alpha-pregnan-3 alpha-ol-11,20-dione, 5 alpha-androstan-3 alpha-ol-17-one, dehydroepiandrosterone sulfate, pregnenolone sulfate and aminosteroid (U-107) were ineffective in that test. Furthermore, 5 alpha-pregnan-3 alpha-ol-20-one (5-20 mg/kg), 5 beta-pregnan-3 alpha-ol-20-one (20 mg/kg) and 5 alpha-androstan-3 alpha-ol 17-one (10 and 20 mg/kg) decreased, while dehydroepiandrosterone sulfate (25 and 50 mg/kg) and pregnenolone sulfate (25 mg/kg) elevated the kainate-induced lethality in mice. A histological analysis showed that kainate caused a dose-dependent neuronal loss of CA1 and CA3 hippocampal fields. Of the neurosteroids tested, only allopregnanolone attenuated the kainate-induced neurotoxicity. The above data indicate that neurosteroids exert moderate effects on seizures and neurotoxic effects of kainate. On the other hand, neurosteroids with a GABAA receptor agonistic or antagonistic activity decrease or increase, respectively, the kainate-evoked lethality.