ENT-A010, a Novel Steroid Derivative, Displays Neuroprotective Functions and Modulates Microglial Responses

Abstract

Tackling neurodegeneration and neuroinflammation is particularly challenging due to the complexity of central nervous system (CNS) disorders, as well as the limited drug accessibility to the brain. The activation of tropomyosin-related kinase A (TRKA) receptor signaling by the nerve growth factor (NGF) or the neurosteroid dehydroepiandrosterone (DHEA) may combat neurodegeneration and regulate microglial function. In the present study, we synthesized a C-17-spiro-cyclopropyl DHEA derivative (ENT-A010), which was capable of activating TRKA. ENT-A010 protected PC12 cells against serum starvation-induced cell death, dorsal root ganglia (DRG) neurons against NGF deprivation-induced apoptosis and hippocampal neurons against Aβ-induced apoptosis. In addition, ENT-A010 pretreatment partially restored homeostatic features of microglia in the hippocampus of lipopolysaccharide (LPS)-treated mice, enhanced Aβ phagocytosis, and increased Ngf expression in microglia in vitro. In conclusion, the small molecule ENT-A010 elicited neuroprotective effects and modulated microglial function, thereby emerging as an interesting compound, which merits further study in the treatment of CNS disorders.

Keywords: Aβ; DHEA; ENT-A010; hippocampus; microglia; neuroprotection; phagocytosis; ΤRKA.

Figure 1

Synthesis of ENT-A010. Reagents and conditions: (a) (CH₃CH₂O)₂P(O)CH₂C(O)OCH₂CH₃, EtONa, THF/EtOH 1:1, reflux, overnight; (b) TBSCl, Imidazole, I_2, THF, 0 °C to 25 °C, overnight; (c) DIBAL-H, CH₂Cl₂, −78 °C, 2.5 h; (d) CH₂I₂, Et₂Zn, −78 °C to 25 °C, 1 h; (e) Dess-Martin periodinane, DCM, 0 °C to 25 °C, 1.5 h; (f) (CH₃CH₂O)₂P(O)CH₂C(O)OCH₂CH₃, NaH, THF, 0 °C to 25 °C, 0.5 h; (g) TBAF (1.0 M in THF), THF, 0 °C to 25 °C, overnight.

Figure 2

ENT-A010 induced TRKA phosphorylation. PC12 cells were treated for 30 min with NGF (100 ng/mL), ENT-A010 (500 nM), or vehicle control. TRKA was immunoprecipitated, and membranes were immunoblotted for phosphor-Tyrosine (pTyr). Whole-cell lysates were analyzed for TRKA. Representative blots are shown. The intensity of the bands was measured, the ratio pTyr/TRKA was calculated and set in each experiment as 1. Data are shown as mean ± SEM, **: p < 0.01; n = 5 independent experiments; ns: non-significant.

Figure 3

ENT-A010 promoted survival of PC12 cells via TRKA. PC12 cells were treated for 24 h with NGF (100 ng/mL), ENT-A010 (500 nM) or vehicle control, with or without the selective TRKA inhibitor GW174456 under serum starvation. Upper panel: Cells were stained with CellTox (green) and Hoechst (blue). Scale bar: 200 μm (applies to all photos). Lower panel: Quantification of cell viability, calculated as percentage of dead (CellTox⁺) cells per total number of Hoechst⁺ cells in each image. Data are shown as mean ± SEM, n = 3–7, **: p < 0.01, ****: p < 0.0001, ns: non-significant.

Figure 4

ENT-A010 promoted DRG neuron survival. DRG neurons were cultured for 2 days in a NGF-free and anti-NGF supplemented medium in the presence of NGF, ENT-A010 or control vehicle. Left panel: Cells stained for TUJ1 (red), with TUNEL (green) or Hoechst (blue). Scale bar: 100 μm (applies to all photos). Right panel: The quantification demonstrates the number of TUNEL⁺ (apoptotic) cells as % of Hoechst⁺ (total) cells. Data are shown as mean ± SEM, n = 4, **: p < 0.01, ***: p < 0.001.

Figure 5

ENT-A010 protected primary hippocampal neurons against oligomeric Aβ toxicity. (A) Primary hippocampal neurons were treated for 48 h with 5 µM oligomeric Aβ and ENT-A010 (500 nM, freshly added every 24 h) or control vehicle and stained for TUJ1 (red), with TUNEL (green) or Hoechst (blue). The quantification shows the number of TUNEL⁺ (apoptotic) cells as % of the total number of cells. (B) Photomicrographs of primary hippocampal neurons, treated for 4 h with 5 µM oligomeric Aβ and ENT-A010 (500 nM) and immunostained against TUJ1 and Synaptophysin. The Synaptophysin- and TUJ1-positive areas were quantified and the ratio (Synaptophysin⁺ area /TUJ1⁺ area) was calculated and normalized in each experiment to the control. Ten to twelve images were acquired per sample. Scale bar: 100 μm (applies to all non-zoomed photos), and 10 μm (applies to all zoomed-in photos). Data are shown as mean ± SEM, n = 3–4, *: p < 0.05, **: p < 0.01.

Figure 6

ENT-A010 induced TRKA and AKT signaling and promoted phagocytosis in microglial cells. (A) Primary microglia cells were treated for 30 min with 1 μM ENT-A010 or DMSO (Ctrl). Cell lysates were analyzed by WB for phospho- and total TRKA (A). (B) BV2 cells were treated for 15, 30 or 60 min with 1 μM ENT-A010 or for 60 min with DMSO (Ctrl) and analyzed for phospho- and total AKT by WB. A representative out of 4 experiments is shown (A,B). Signal intensities of pTRKA, total TRKA, pAKT and total AKT were measured and in each experiment, the ratio of pTRKA/TRKA and pAKT/AKT was set as 1 for Ctrl samples. (B) shows the quantification of pAKT/AKT at 60 min of treatment. (C,D) Primary microglia were treated with 1 μM ENT-A010 on two consecutive days, and 1 h after the second treatment, they were stimulated with 100 ng/mL LPS. In (D), cells were pre-treated with the AKT inhibitor MK2206 (2.5 µM) 30 min prior to the first ENT-A010 treatment. Twenty-four h after the LPS treatment, fluorescently labeled Aβ was applied for 2 h to the cells, followed by flow cytometry. Data are shown as mean ± SEM, n = 4 for (A), n = 4 for (B), n = 6 (C), n = 4–10 (D), *: p < 0.05, **: p < 0.01, ns: non-significant.

Figure 7

ENT-A010 promotes a protective microglial phenotype in vitro. Primary microglia were treated with 1 μM ENT-A010 on two consecutive days, and 1 h after the second treatment, they were stimulated with 100 ng/mL LPS. The expression of indicated genes was analyzed by qPCR. Data are shown as mean± SEM n = 25 for (A), n = 19 for (B), n = 10 for (C–E) *: p < 0.05.

Figure 8

Peripherally administered ENT-A010 is detected in the brain. Mice were i.p. injected with 70 mg/kg ENT-A010 and 1 and 2 h later indicated brain regions, the liver and the spleen were collected, snap-frozen and analyzed by UHPLC-MS. Control mice were injected with the same amount of carrier solution (4.5% ethanol, 1% BSA, PBS). Data are shown as mean ± SEM, n = 3 mice per condition, *: adj p < 0.05 **: adj p < 0.01 ***: adj p < 0.005 ****: adj p < 0.0001.

Figure 9

ENT-A010 restores microglial homeostatic features in the hippocampus of LPS-treated mice. (A) Mice were i.p. injected with 70 mg/kg ENT-A010 or control solution on two consecutive days. One h after the second treatment, they received i.p. 3 mg/kg LPS. After 16 h whole brains were isolated and immunostained against IBA1 (red) and with DAPI (blue). The whole hippocampal formation was imaged. Representative photomicrographs partially include the CA1 region, stratum radiatum and pyramidal layer (upper panel)—scale bars: 50 µm. (B–M). Hippocampi were isolated from mice treated as described in (A) and the whole RNA was analyzed by real-time PCR for indicated genes, using 18S as a housekeeping gene. Fold change of relative gene expression was calculated based on the gene expression in the ‘LPS’ samples. Data are shown as mean ± SEM, n = 3–10 mice, *: p < 0.05, **: p < 0.01, ns: non-significant.