Stereospecific Properties and Intracellular Transport of Novel Intrinsically Fluorescent Neurosteroids

Abstract

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABA_A) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well. We found stereospecific differences in the orientation and dynamics of the NAS as well as in their impact on membrane permeability. However, all NAS are unable to condense the lipid bilayer, in stark contrast to cholesterol. Using Förster resonance energy transfer (FRET) and electrophysiological measurements, we show that MQ-323 but not YX-11 binds at the intersubunit site of the ELICα₁GABA_A receptor and potentiates GABA-induced receptor currents. In aqueous solvents, YX-11 forms aggregates at much lower concentrations than MQ-323, and loading both analogs onto cyclodextrin allows for their uptake by human astrocytes, where they become enriched in lipid droplets (LDs), as shown by quantitative fluorescence microscopy. Trafficking of the NAS analogs is stereospecific, as uptake and lipid droplet targeting is more pronounced for YX-11 compared to MQ-323. In summary, we present novel minimally modified analogs of AlloP and E-AlloP, which enable us to reveal stereospecific membrane properties, allosteric receptor activation, and intracellular transport of these neurosteroids. Our fluorescence design strategy will be very useful for the analysis of other NAS in the future.

Keywords: allopregnanolone; astrocytes; fluorescence; microscopy; probes; trafficking.

Figure 1:

Allopregnanolone enantiomers and their fluorescent analogs can order phospholipid acyl chains. Chemical structures of allopregnanolone and epi-Allopregnalone (A) and their fluorescent analogs YX-11 and MQ-323 (B), respectively. The structure of allopregnanolone and epi-allopregnanolone differ from each other only by the orientation of the 3-hydroxyl group (highlighted in green and red, respectively). The fluorescent properties of the allopregnanolone and epi-allopregnanolone analogous, named YX-11 and MQ-323, are provided by the conjugated double bonds (highlighted in violet). Acyl chain order parameter calculated from MD trajectories for the sn-1 chain (palmitic acid, PA in panel C) and the sn-2 chain (oleic acid, OA in panel D) for membranes consisting of POPC and the indicated combinations of cholesterol (CHL) and neurosteroids.

Figure 2:

Membrane localization and orientation of allopregnanolone enantiomers (A-B) and their fluorescent analogs (C-D) and cholesterol (E). Density heatmaps (2D-histograms) over the tilt angle and distance from the membrane center (z) are shown.

Figure 3:

Hydrogen bonding capacity and membrane dynamics of allopregnanolone enantiomers and their fluorescent analogs. The number of hydrogen bonds formed between the allopregnanolone enantiomers and water (blue lines), the POPC carbonyl groups (orange lines), and cholesterol’s hydroxyl group (green lines) are shown for all neurosteroids (A-D). The black lines indicate the total number of hydrogen bonds formed by membrane-embedded neurosteroids. Membrane dynamics of neurosteroids were assessed in relation to that of cholesterol from the decay of the autocorrelation function as a function of time, ACF(t), as shown in (E). Panels (F-D) show snapshots from the MD simulations of MQ-323 and YX-11. The neurosteroid molecules are highlighted as spheres.

Figure 4:

Order parameter of neurosteroids and their effect on membrane permeability to dithionite. The order parameter of the sn-1 chain of deuterated POPC in the indicated membranes was measured by ²H-NMR spectroscopy (A-C). Permeability of large unilamellar vesicles consisting of the indicated lipids and containing 0.5 mol% NBD-PC to sodium dithionite was measured by fluorescence spectroscopy (D). A bi-exponential fit to the data gives the rate constant for access of dithionite to the inner membrane leaflet.

Figure 5:

Förster resonance energy transfer (FRET) of neurosteroid binding to GABAa receptors. Purified ELICα₁GABA_A receptor was incubated with either MQ-323 or YX-11 dissolved in a buffer containing 0.015% DDM, and FRET from the protein to the respective NAS was measured upon excitation at 280 nm. Raw emission spectra (A) and normalized FRET intensities (B, n = 3 for each plot) for increasing concentrations of MQ-323. Nonspecific binding in (B) is the FRET signal obtained in the presence of 30 μM allopregnanolone, and W246L shows that the FRET is reduced to non-specific binding when W246 in the receptor is replaced by leucine. Comparison of total and non-specific FRET obtained with MQ-323 and YX-11 (C), showing that YX-11 does not bind to the ELICα₁GABA_A receptor.

Figure 6:

Electrophysiological measurements of GABA_A receptor currents. The α1β3γ2L receptors were activated by 2 μM GABA (A, B) or 1 mM GABA + 50 μM propofol (C), and modulated by 10 μM MQ-323 or YX-11. Drug applications are shown by horizontal lines above current traces. The dot plots show steroid effects in % of control response (100% = no effect) calculated as ratios of current amplitudes after and before application of steroid.

Figure 7:

Fluorescence emission spectra of YX-11 and MQ-323 titration in water. (A+B) Normalized fluorescence emission spectra (Ex. 328nm) of YX-11 and MQ-323 with various concentrations (0 to 50 μM) in water. The spectra represent the average from three independent measurements and are normalized to the maximum peak intensity. (C) The mean of the maximum emission at 416 nm and 378 nm from three individual experiments of YX-11 (orange) and MQ-323 (blue), respectively, plotted as a function of neurosteroid concentration and fitted with a straight-line model with breakpoints using a piece-wise linear regression routine.

Figure 8:

Uptake of neurosteroid analogs loaded on methyl-β-cyclodextrin (MCβD) by astrocytes. Human astrocytes were loaded with either YX-11 and MQ-323 on MCβD (final concentration of 20 μM) and incubated for 1 hr before imaging on a UV-sensitive wide-field microscope (A-B). Scale bar, 20 μm. Cells were segmented using Cellpose (see Image analysis for fluorescence microscopy data) in order to find the mean UV fluorescence intensity for cells loaded with YX-11 (n = 580) or MQ-323 (n = 488) (C). Control in panel C are cells without loading of neurosteriods (n = 290). By uing a Mann-Whitney U test on the distributions for YX-11 and MQ-323 in panel C, a p-value of 9.510 × 10⁻³² was found.

Figure 9:

Neurosteriods are localized in lipid droplets (LDs). Human astrocytes were loaded with either YX-11 or MQ-323 on MCβD (final concentration of 20 μM), incubated for 1 hr and afterward stained with bodipy for 5 min before imaging on a UV-sensitive wide-field microscope (A-B). Scale bar, 20 μm. Cells and LDs were segmented as described in Image analysis for fluorescence microscopy datas, and the fraction of YX-11 (n = 290) or MQ-323 (n = 223) found inside LDs per cell was quantified (C). A fraction above 1 indicates the upregulation of neurosteroids in LDs. By using a Mann-Whitney U test on the distributions in panel C, a p-value of 7.66 × 10⁻³⁰ was found.