A.G. Mensah-Nyagan

Potential role of allopregnanolone for a safe and effective therapy of neuropathic pain.

Because the treatment and management of neuropathic pain are extremely complicated, the characterization of novel analgesics and neuroprotectors with safe toxicological profiles is a crucial need to develop efficient therapies. Several investigations revealed that the natural neurosteroid allopregnanolone (AP) exerts analgesic, neuroprotective, antidepressant and anxiolytic effects. These effects result from AP ability to modulate GABA(A), glycine, L- and T-type calcium channels. It has been shown that AP treatment induced beneficial actions in humans and animal models with no toxic side effects. In particular, a multi-parametric analysis revealed that AP efficiently counteracted chemotherapy-evoked neuropathic pain in rats. It has also been demonstrated that the modulation of AP-producing enzyme, 3α-hydroxysteroid oxido-reductase (3α-HSOR), in the spinal cord regulates thermal and mechanical pain thresholds of peripheral nerve injured neuropathic rats. The painful symptoms were exacerbated by intrathecal injections of provera (pharmacological inhibitor of 3α-HSOR) which decreased AP production in the spinal cord. By contrast, the enhancement of AP concentration in the intrathecal space induced analgesia and suppression of neuropathic symptoms. Moreover, in vivo siRNA-knockdown of 3α-HSOR expression in healthy rat dorsal root ganglia increased thermal and mechanical pain perceptions while AP evoked a potent antinociceptive action. In humans, blood levels of AP were inversely associated with low back and chest pain. Furthermore, oral administration of AP analogs induced antinociception. Altogether, these data indicate that AP, which possesses a high therapeutic potential and a good toxicological profile, may be used to develop effective and safe strategies against chronic neuropathic pain.

Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

Allopregnanolone and its analog BR 297 rescue neuronal cells from oxidative stress-induced death through bioenergetic improvement

Allopregnanolone (AP) is supposed to exert beneficial actions including anxiolysis, analgesia, neurogenesis and neuroprotection. However, although mitochondrial dysfunctions are evidenced in neurodegenerative diseases, AP actions against neurodegeneration-induced mitochondrial deficits have never been investigated. Also, the therapeutic exploitation of AP is limited by its difficulty to pass the liver and its rapid clearance after sulfation or glucuronidation of its 3-hydroxyl group. Therefore, the characterization of novel potent neuroprotective analogs of AP may be of great interest. Thus, we synthesized a set of AP analogs (ANS) and investigated their ability to counteract APP-overexpression-evoked bioenergetic deficits and to protect against oxidative stress-induced death of control and APP-transfected SH-SY5Y cells known as a reliable cellular model of Alzheimers disease (AD). Especially, we examined whether ANS were more efficient than AP to reduce mitochondrial dysfunctions or bioenergetic decrease leading to neuronal cell death. Our results showed that the ANS BR 297 exhibits notable advantages over AP with regards to both protection of mitochondrial functions and reduction of oxidative stress. Indeed, under physiological conditions, BR 297 does not promote cell proliferation but efficiently ameliorates the bioenergetics by increasing cellular ATP level and mitochondrial respiration. Under oxidative stress situations, BR 297 treatment, which decreases ROS levels, improves mitochondrial respiration and cell survival, appears more potent than AP to protect control and APP-transfected cells against H2O2-induced death. Our findings lend further support to the neuroprotective effects of BR 297 emphasizing this analog as a promising therapeutic tool to counteract age- and AD-related bioenergetic deficits.

Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations <50 microM. Above this dose, GHB effects were inactivated. In addition, at GHB concentrations exceeding 50 microM, GTP-gammaS binding was also reduced, confirming the desensitization of GHB receptor(s). Taken together, these results support the existence in NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes.

Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic‐pituitary‐adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP‐based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell‐proliferation‐promoting effects, hamper the development of selective/targeted therapies. For example, although AP‐induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP‐evoked cell‐proliferation is contraindicated for steroid‐sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12‐oxo‐epi‐AP), BR297 (O‐allyl‐epi‐AP), BR351 (O‐allyl‐AP) and BR338 (12‐oxo‐AP). First, because AP is well‐known as allosteric modulator of GABAA receptors (GABAA‐R), we used the electrophysiological patch‐clamp technique to determine the structure‐activity relationship of our ANS on GABAA‐activated current in NCB20 cells expressing functional GABAA‐R. We found that the addition of 12‐oxo‐group did not significantly change the respective positive or negative allosteric effects of 3α‐AP or 3β‐(epi)‐AP analogues. Importantly, substitution of the 3α‐hydroxyl‐group by 3α‐O‐allyl highly modified the ANS activities. Unlike AP, BR351 induced a long‐lasting desensitisation/inhibition of GABAA‐R. Interestingly, replacement of the 3β‐hydroxyl by 3β‐O‐allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH‐SY5Y neuronal cell viability/proliferation using MTT‐reduction assays. Different dose‐response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell‐proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress‐induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH‐SY5Y cells against oxidative stress‐induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell‐proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid‐based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

Protective effect of 4-Phenylbutyrate against proteolipid protein mutation-induced endoplasmic reticulum stress and oligodendroglial cell death

ABSTRACT Proteolipid protein (PLP) mutation causes oligodendrocyte degeneration and myelin disorders including Pelizaeus‐Merzbacher Disease (PMD). As the pathophysiological mechanisms involved in PMD are poorly known, the development of therapies remains difficult. To elucidate the pathogenic pathways, an immortalized oligodendroglial cell line (158JP) expressing PLP mutation has been generated. Previous investigations revealed that 158JP oligodendrocytes exhibit several abnormalities including aberrant PLP insertion into the plasma membrane, cAMP, plasmalogen and cell cycle deficits. However, further clarifications of abnormal PLP‐induced oligodendrocyte degeneration are required in order to identify relevant mechanisms to target for efficient protection against oligodendrocyte death. Because PLP overexpression may lead to its accumulation inside the endoplasmic reticulum (ER) and cause ER‐stress, we explored whether ER‐stress may pivotally determine 158JP cell survival/death. Viability assays, RT‐qPCR, western blot and flow cytometry were combined to compare cell survival, ER‐stress and apoptotic markers in 158JP and control (158N) oligodendrocytes. We observed a significant decreased viability/survival of 158JP compared to 158N cells. Consistently, ER‐stress markers (BiP, caspase‐12) increased in 158JP (+30%) compared to the controls. mRNA and protein ratios of apoptotic modulators (Bax/Bcl2) are higher in 158JP oligodendrocytes which are also more vulnerable than 158N cells to tunicamycin‐induced ER‐stress. Interestingly, 4‐Phenylbutyrate (ER‐stress inhibitor), which decreased ER‐stress and apoptotic markers in 158JP cells, significantly increased their survival. Our results, which show a direct link between the viability and endogenous levels of ER‐stress and apoptotic markers in 158JP cells, also suggest that 4‐Phenylbutyrate‐based strategy may contribute to develop effective strategies against oligodendrocyte dysfunctions/death and myelin disorders. Highlights158JP oligodendrocytes harboring PLP mutation are less viable than normal 158N cells.Basal levels of ER stress and apoptosis markers are higher in 158JP than 158N cells.158JP cells are more vulnerable to tunicamycin‐evoked ER‐stress than 158N cells.ER stress pivotally determines PLP mutation‐induced 158JP oligodendrocyte death.4‐PBA which reduces ER‐stress and apoptotic levels protects 158JP cells against death.

The translocator protein ligand XBD173 improves clinical symptoms and neuropathological markers in the SJL/J mouse model of multiple sclerosis

Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory, demyelinating and neurodegenerative components causing motor, sensory, visual and/or cognitive symptoms. The relapsing-remitting MS affecting 85% of patients is reliably mimicked by the proteolipid-protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) SJL/J-mouse model. Significant progress was made for MS treatment but the development of effective therapies devoid of severe side-effects remains a great challenge. Here, we combine clinical, behavioral, histopathological, biochemical and molecular approaches to demonstrate that low and well tolerated doses (10-20mg/kg) of TSPO ligand XBD173 (Emapunil) efficiently ameliorate clinical signs and neuropathology of PLP-EAE mice. In addition to the conventional clinical scoring of symptoms, we applied the robust behavioral Catwalk-method to confirm that XBD173 (10mg/kg) increases the maximum contact area parameter at EAE-disease peak, indicating an improvement/recovery of motor functions. Consistently, histopathological studies coupled with microscope-cellSens quantification and RT-qPCR analyzes showed that XBD173 prevented demyelination by restoring normal protein and mRNA levels of myelin basic protein that was significantly repressed in PLP-EAE mice spinal cord and brain. Interestingly, ELISA-based measurement revealed that XBD173 increased allopregnanolone concentrations in PLP-EAE mice spinal and brain tissues. Furthermore, flow cytometry assessment demonstrated that XBD173 therapy decreased serum level of pro-inflammatory cytokines, including interleukin-17A, Interleukin-6 and tumor-necrosis-factor alpha in PLP-EAE mice. As the optimal XBD173 dosing exerting the maximal beneficial action in EAE mice is the lower 10mg/kg dose, the paper opens interesting perspectives for the development of efficient and safe therapies against MS with slight or no side-effects.