Sex-dependent neuronal effects of α-synuclein reveal that GABAergic transmission is neuroprotective of sleep-controlling neurons
Cell & Bioscience volume 13, Article number: 172 (2023)
Sleep disorders (SDs) are a symptom of the prodromal phase of neurodegenerative disorders that are mechanistically linked to the protein α-synuclein (α-syn) including Parkinson’s disease (PD). SDs during the prodromal phase could result from neurodegeneration induced in state-controlling neurons by accumulation of α-syn predominant early in the disease, and consistent with this, we reported the monomeric form of α-syn (monomeric α-syn; α-synM) caused cell death in the laterodorsal tegmental nucleus (LDT), which controls arousal as well as the sleep and wakefulness state. However, we only examined the male LDT, and since sex is considered a risk factor for the development of α-syn-related diseases including prodromal SDs, the possibility exists of sex-based differences in α-synM effects. Accordingly, we examined the hypothesis that α-synM exerts differential effects on membrane excitability, intracellular calcium, and cell viability in the LDT of females compared to males.
Patch clamp electrophysiology, bulk load calcium imaging, and cell death histochemistry were used in LDT brain slices to monitor responses to α-synM and effects of GABA receptor acting agents.
Consistent with our hypothesis, we found differing effects of α-synM on female LDT neurons when compared to male. In females, α-synM induced a decrease in membrane excitability and heightened reductions in intracellular calcium, which were reliant on functional inhibitory acid transmission, as well as decreased the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) with a concurrent reduction in action potential firing rate. Cell viability studies showed higher α-synM-mediated neurodegeneration in males compared to females that depended on inhibitory amino acid transmission. Further, presence of GABA receptor agonists was associated with reduced cell death in males.
When taken together, we conclude that α-synM induces a sex-dependent effect on LDT neurons involving a GABA receptor-mediated mechanism that is neuroprotective. Understanding the potential sex differences in neurodegenerative processes, especially those occurring early in the disease, could enable implementation of sex-based strategies to identify prodromal PD cases, and promote efforts to illuminate new directions for tailored treatment and management of PD.
Parkinson’s disease (PD) is among one of the most widespread neurodegenerative disorders [1, 2], and sex is a risk-factor in the development of this disease, as PD is more common in men than in women with an approximated odds ratio of 2:1 [3,4,5,6,7]. Although PD is clinically diagnosed by the cardinal motor symptoms, evidence emerging over the past two decades has established sleep disorders (SDs) such as REM sleep behavior disorder (RBD), which is a sleeping disorder characterized by excessive motor behavior during what is normally a period of atonia, and excessive daytime sleepiness (EDS) as markers of the prodromal phase of PD, and these SDs can precede the motor symptoms by years to decades [8,9,10,11,12,13]. Sex has been acknowledged as an important determinant of both the susceptibility to neurodegenerative diseases and whether SDs co-occur following diagnosis, and while not well studied, it is likely that sex differences are also present in the prodromal phase of the disease prior to diagnosis, which could include the expression of SDs.
We hypothesized that the appearance of SDs prodromal to the motor symptoms in PD could be due to alteration of cellular function and neurodegeneration in sleep controlling nuclei and that sex differences in cellular effects could be present. Neurodegeneration in several brain nuclei in PD is associated with aggregation of the protein α-synuclein (α-syn), which is the histological hallmark of PD . Pathological studies have shown that aggregated α-syn can interfere with several cellular functions in addition to promoting cell toxicity . Heightened cell death has been reported in patients with α-synucleinopathies in the laterodorsal tegmentum (LDT) , which is a heterogenous nucleus comprised of cholinergic, glutamatergic and GABAergic neurons  that are importantly involved in the control of motor atonia during sleep, and arousal during wakefulness [18, 19]. Further, the co-occurrence of RBD and α-syn pathology was strongly correlated with brainstem cholinergic dysfunction in a predominantly male cohort consistent with α-syn-mediated degeneration of LDT cholinergic systems underlying aberrant behavioral state behavior . Taken together, this suggests that α-syn-mediated PD processes include cellular actions in SD-controlling neurons already from abnormal levels of α-syn.
Although several studies have investigated the neuronal effects induced by α-syn, their focus was on actions of forms of the protein which aggregate (oligomeric and fibril). These forms are suspected to be the most damaging to neurons and neural transmission, and so very few studies have reported on cellular effects induced by the disordered, native monomeric form (α-synM), which is widely considered to be relatively benign. The aggregation and fibrillation of α-syn may be preceded by dysregulation of expression e.g. as a result of SNCA gene multiplication ) and/or clearance  leading to abnormal levels of α-syn in the CNS, which then in turn may lead to nucleation and fibril formation . We recently found that α-synM has cellular effects on neurons in the LDT that were associated with heightened cell death, which we speculated could play a role in SDs prodromal to, as well as following, diagnosis of PD. Specifically, we found that α-syn in monomeric form induced excitation, increased intracellular calcium, and heightened neuronal death of neurons of the LDT. In contrast, different effects were induced in the substantia nigra (SN) in that α-synM elicited membrane inhibition, and greater decreases of intracellular calcium with no evidence of α-synM-induced cell death . However, that investigation was conducted solely in LDT of males. Because the appearance of sex-based differences in α-syn-related disease symptoms, including differences between SDs, suggest different mechanistic actions are involved in disease processes in males and females, we wished to determine whether excitatory cellular effects of α-synM on LDT, and heightened cell death in males were also present in females. Accordingly, in the present report, we have used electrophysiological and calcium imaging techniques ex vivo to investigate cellular effects of highly purified α-synM on LDT and SN neurons from females and compared effects to those in male LDT neurons.
The protocols for animal experiments used in this study were approved in concordance with the European Communities Council Directive (86/609/EEC). Brain slices of 250 μm thickness from female and male Naval Medical Research Institute (NMRI) mice aged 12 to 30 days (Harlan Mice Laboratories, Denmark) were used in electrophysiological, calcium imaging and cell viability studies. The animals were housed under the following conditions: temperature (22–23 °C), humidity (45–65%), light-dark cycle 12:12 h, water and food were available ad libitum.
Brain slice preparations
A state of anesthesia was induced via inhalation of isoflurane (Baxter A/S, Denmark) and decapitation was conducted when anesthesia had been achieved as assayed by failure to react to a paw pinch. A block of the brain which contained LDT or SN was rapidly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF). The ACSF solution which contained 124 NaCl, 5 KCl, 1.2 Na2HPO4•2H2O, 2.7 CaCl2•2H2O, 1.2 MgSO4 (anhydrous), 10 dextrose, 26 NaHCO3 in mM was adjusted to a pH of 7.4 and an osmolarity of 298–302 mOsm/kg following saturation with carbogen (95% O2/5% CO2). The brain was sectioned in 250 μm thick slices containing the LDT or the SN with a vibratome (Leica VT1200S, Leica Biosystems, Germany). Brain slices were collected and placed in a chamber containing oxygenated ACSF, and incubated at 37 oC for 15 min. To allow the tissue to equilibrate after the incubation period, the slices were kept at room temperature, and carbogen was continuously supplied for at least 1 h prior to further procedures, including exposure of the slice to the monomeric form of α-syn.
Human α-syn was recombinantly expressed and purified as described in more detail in our earlier published work using this peptide . Briefly, α-syn was cloned into E. Coli BL21DE3 cells using a pET-11a vector construct. Harvested cells were lysed by osmotic shock. Subsequently, boiling and centrifugation were conducted to remove non-heat-stable proteins. Ion-exchange chromatography was used to isolate α-syn, and the monomeric fraction was isolated by size exclusion chromatography SEC; thereafter, the monomers were pooled and kept in PBS buffer stored at -80oC until application to the slices.
The monomeric form of α-syn (α-synM) was stored in solution at -20oC in aliquots of 10 µl (150 µM) until use at which time it was applied via the bath. To reach a final concentration of 100 nM, an aliquot (150 µM) of 10 µl of α-synM was diluted in ACSF. After the establishment of baseline holding currents or baseline fluorescence, α-synM was applied for 3–4 min to monitor effects on membrane holding currents, synaptic activity, action potential firing, and intracellular calcium. For cell viability studies, incubation of the 250 μm brain slice in α-synM for 7 h was conducted in protocols described below.
Action potentials within the slice were blocked by 0.5 mM tetrodotoxin (TTX, Tocris, UK). Glycinergic receptors were blocked with strychnine (2.5 µM; Sigma, Denmark). GABAA and GABAB receptor-mediated responses were blocked by SR-95,531 (gabazine, 10 µM, Sigma, Demark) and CGP 55,845 (10 µM, Tocris, UK), respectively. Muscimol (30 µM, Sigma, Denmark) and baclofen (10 µM, Sigma, Denmark) were used as agonists of the GABAA and GABAB receptors, respectively. Stock solutions were stored in appropriate aliquots at -20oC prior and diluted in ACSF to final concentrations before use, and all drugs were applied via the bath.
Patch-clamp recordings to monitor changes in membrane currents, synaptic activity, and action potential firing
Whole cell patch clamp recordings were conducted in 250 μm thick brain slices from neurons in the LDT (19 LDT brain slices; 14 mice) or the SN (7 SN brain slices; 6 mice). For recordings done in the LDT, we wished to target cholinergic neurons. Therefore, we selected the recorded neurons based on soma size (medium-to-large cells) and location within the central LDT wherein the concentration of cholinergic neurons is highest . To fabricate patch pipette electrodes for recording neuronal electrical activity in LDT and SN brain slices, borosilicate filamented glass capillary tubes (1.5 mm, Sutter Instruments, USA) were pulled after heating to a sharp tip in a horizontal Flaming/Brown micropipette puller (P-97, Sutter Instruments, USA). These glass electrodes were filled with an intracellular solution (144 K-gluconate; 2 KCl; 10 HEPES; 0.2 EGTA; 5 Mg-ATP and 0.3 Na-GTP; in mM), which resulted in a pipette resistance of 6–11 MΩ. A brain slice containing LDT or SN was placed in the recording chamber that was situated in a microscope stage and ACSF saturated with a mixture of 95% oxygen/5% carbon dioxide (carbogen) was continuously perfused over the slice (flow rate 1.2 mL/min). A water immersion objective (60x) coupled to an upright microscope (BX50WI, Olympus; Japan) with an infrared Dodt gradient contrast system (IR-DGC; Luigs & Neumann, Germany) and a CCD camera (CCD-300ETRC; DAGE-MTI, Michigan City, IN) were used to visualize the cells. The software, Patchmaster (HEKA; version v2 × 91), was used to control a patch-clamp EPC9 amplifier (HEKA, Germany). Recordings were initiated in voltage-clamp mode to establish high resistance seals (> 1 MΩ) between the patch pipette and the cell membrane, and the holding voltage was kept at -60 mV. After at least a stabilization period of 7 min following membrane breakthrough, data were collected. AxoScope 10.2 (Molecular Devices Corporation, USA) and an Axon miniDigi 1B digitizer (Molecular Devices Corporation) were used to sample membrane effects. To quantify relative changes in holding currents, the holding currents in pA averaged from at least 30 sec of recording before application of α-synM and the holding currents averaged from at least 30 sec during the maximum effect of α-synM were subtracted. A change in amplitude of 2 pA from baseline was used as criteria to be considered a response. For firing frequency studies, action potentials were recorded in current-clamp mode before and after α-synM application. Current was applied to depolarize the neuron sufficiently to induce a sustained firing of action potentials (-45 mV). A period of firing in an epoch of 30 sec immediately prior to and 30 sec after application of α-synM was selected and interspike intervals were measured and averaged. Interspike intervals were determined by measuring the period of time (in msec) from the initiation of a spike to initiation of the next spike.
Calcium imaging to monitor changes in intracellular calcium
Intracellular loading of cells with a calcium binding dye was performed in 42 LDTF (25 mice) and 15 LDTM (7 mice) 250 μm thick brain slices following standard protocols  for single-photon calcium imaging. Recordings were conducted utilizing ratiometric fluorescent calcium indicator dye Fura-2 acetoxymethyl ester (Fura-2 AM). Prior to recordings, slices were rinsed for over 15 min in the recording chamber by continuous perfusion of oxygenated ACSF at a flow rate of 1.2 ml/min to wash out free dye debris and allow temperature equilibration. To localize the LDT, an upright microscope (BX50WI, Olympus, Germany) was used under bright field illumination and visualization guided by characteristic landmarks located close to these nuclei. Individual cells were viewed with a water immersion objective (40x). A cooled CCD fluorescence camera (12-bit Sensicam, PCO Imaging, Germany) attached to the microscope and controlled by the imaging software Live Acquisition (TILL Photonics, Germany) was used to collect paired images that were binned at 2 × 2 pixels on the camera chip in order to optimize spatial and temporal resolution. Live Acquisition controlled rapid switching between the excitation wavelengths of 340 and 380 nm in order to ensure a minimal amount of time passing between collection of the image at each wavelength, which allowed ratiometric calculations. The acquisition interval between each frame pair of 340 and 380 nm was 4 sec. Regions of interest (ROI) were drawn around each Fura-2 loaded cell, as well as around a region of the field of view that contained no dye loaded cells, which was used to measure background fluorescence. Analysis software (Offline Analysis, TILL Photonics, Germany) was used to quantify fluorescence in each ROI by averaging the intensity of the 2 × 2 binned pixels, and background in each channel was subtracted. The 340 and 380 nm channels were ratioed (340 nm:380 nm). Baseline fluorescence was calculated from an average of intensity taken from 10 frames collected before drug application. Following application of α-synM, changes in fluorescence from that measured at baseline were noted in the majority of cells, and the fluorescence of the peak deflection from baseline was measured by averaging across 10 frames. Data are presented as DF/F% which represents the subtraction of the baseline fluorescence from the maximum change in fluorescence induced by α-synM application divided by the baseline, followed by conversion to a percentage. Ascendent deflection of fluorescence indicates intracellular calcium elevations with descendent deflections reflecting decreases in intracellular calcium. Actual calcium levels were not calculated due to well-known complications with converting changes in fluorescence in brain slices to calcium concentrations [26, 27].
Neurotoxicity assay to evaluate cell viability
Propidium iodide (PI; Sigma-Aldrich) was used to identify dead cells and 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used to mark live cells from LDTF and LDTM slices. A total of 116 slices from 56 mice were used in 3 different protocols in which 250 μm slices were bisected with each half being exposed for 7 h under oxygenation to: (1) ACSF or α-synM (100 nM), (2) α-synM (100 nM) or α-synM (100 nM) plus GABAA, GABAB and glycine receptor antagonists, or (3) α-synM (100 nM) or α-synM (100 nM) plus GABAA, GABAB, and glycine receptor agonists. The bisection of the slices ensured that data sets could be compared from tissue taken from the same group of animals, and that protocols were run side by side under the same laboratory conditions. Following incubation, slices were fixed in 4% paraformaldehyde overnight, cryprotected with sucrose saturation (30%), and resectioned to a thickness of 40 μm on a cryostat (Leica CM3050, Triolab, DK). Resectioned slices of 40 μm were incubated for 3 periods of 5 min in a solution which contained 1 µg/ml of both PI and DAPI with a pH of 7.4. To detect fluorescence signals from PI and DAPI, an upright Zeiss microscope coupled to a monochrome CCD camera (Axiocam MRM, Zeiss, Germany) controlled by Axioskop 2 software (AxioVision 4.6, Zeiss) and required filter cubes were used (Zeiss 59 fluorescent filter cube sets, wavelengths PI: 472–578 nm; DAPI: 358–463 nm).
To conduct analysis of collected images, a macro written for ImageJ (National Institutes of Health, Bethesda, MD) was used to automatically count the number of DAPI and PI-labeled cells following background subtraction, application of thresholding, and separation of objects via a watershed algorithm. Cells were selected using the batch processing macro, Analyze Particle; however, selections were manually confirmed. Cell survival was quantified by the proportion of live cells (DAPI positive cells) to the total cell count, which was calculated as the addition of PI positive cells and DAPI positive cells. Cell viability was evaluated in up to 5 areas within the LDT in the 40 μm resectioned slices. For this data set, na = the number of areas examined/n40 = the number of resectioned slices. Data was normalized in each of the 3 protocols to the control condition, which was either exposure to ACSF or α-synM without GABAA, GABAB, and glycine receptor agonists or antagonists. However, to compare cell death between LDTF and LDTM, cell survival was evaluated without normalization. In figure panels depicting PI positive and DAPI positive cells, contrast has been applied equally across entire images.
Data analysis and statistics
Amplitudes of membrane holding currents were measured (the difference between baseline and maximum deflection) using Axoscope 10.5 (Molecular Devices, USA). Spontaneous excitatory synaptic events (sEPSCs) were detected and analyzed using MiniAnalysis (Synaptosoft, USA). Analysis of synaptic events was conducted by selecting 30 sec of the recording just before application of α-synM and at the peak amplitude of the effect on membrane current. Inter-event intervals and the amplitude of events were averaged across a population of cells and statistically analyzed. Firing frequency was analyzed selecting 30 sec epochs before and after α-synM application, and intervals between action potentials were measured and averaged. Calcium imaging data including the amplitudes of DF/F% changes were analyzed in Graphpad Prism (version 7.0). The numbers of observations in the data sets analyzed for cell viability are reflected as na. Results are presented as mean values ± SEM. The figures were prepared using Igor Pro software (Wavemetrics, USA) and GraphPad Prism. Differences in numerical data were tested using a Paired or Unpaired Student’s T-test, and differences in categorical data were examined using the Fisher’s Exact test. P values are reported in text as 4 decimal points, and a significant difference was determined if alpha was less than 0.05.
α-syn M effects on membrane currents, and synaptic transmission in neurons of sleep and motor controlling nuclei in the female
α-synM induced outward currents in sleep and motor control nuclei in the female LDT
Our previous study showed that α-synM induced an inward current in LDT neurons in brain slices from male mice (LDTM) . Unexpectedly, in neurons in LDT brain slices from female mice (LDTF; Fig. 1A), α-synM (100 nM, 3 min) induced an outward membrane current in all cells examined (amplitude: 54.8 ± 11.3 pA, n = 14). To compare the effect of α-synM in neurons of a sleep controlling nucleus vs. neurons of a motor control nucleus, we next investigated the neuronal effect of α-synM in the SN of females (SNF). Similar to our previous report that showed α-synM induced inhibitory membrane responses in neurons of the male SN (SNM), we observed that α-synM induced inhibitory responses in the membrane of 100% of the neurons examined in SNF (amplitude: 47.2 ± 16.0 pA, n = 11). The average amplitude of the outward current induced in neurons of the LDTF did not differ from that induced in SNF neurons (p = 0.3847; Unpaired Student’s T-test; Fig. 1B).
α-synM alters synaptic events in sleep and motor control nuclei in the female LDT
Our previous study showed that α-synM altered synaptic transmission in LDTM neurons producing increases in frequency as well as amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). However, in the present study, the opposite effect was seen in LDTF as α-synM induced a significant decrease of nearly 20% in sEPSC frequency (Control: 7.2 ± 2.7 Hz; α-synM: 5.8 ± 2.3 Hz; p = 0.0475; n = 5, Paired T-test) and a 21% decrease in the amplitude of sEPSCs was noted which was significant (control: 10.8 ± 1.7 pA; α-synM: 8.5 ± 1.3 pA; n = 5; p = 0.0253; Paired T-test; Fig. 1C).
In SNF, α-synM induced a decrease of 14% in amplitude of sEPSCs which was significant when compared to control (control: 6.1 ± 0.8pA; α-synM: 5.2 ± 0.5pA; n = 4; p = 0.0483; Paired T-test); however, changes induced in the frequency were not significantly different (control: 5.9 ± 1.9 Hz; α-synM: 7.4 ± 2.8 Hz; n = 4; p = 0.4735; Paired T-test; Fig. 1C). In our previous work, we did not examine the effect of α-synM on sEPSCs in the SN in the male (SNM). Therefore, in order to examine sex-based potential differences of α-synM on sEPSCs in this motor control nucleus, we determined whether α-synM had effects on synaptic activity in the SNM and found that changes in sEPSCs were qualitatively similar to those seen in SNF. α-synM induced a decrease of 10% in the amplitude of the current of the sEPSCs (Ctrl: 6.0 ± 1.7 pA; α-synM: 5.4 ± 1.6 pA; n = 4; p = 0.0449; Paired T-test), and changes induced in the frequency were not significantly different (Ctrl: 11.8 ± 2.2 Hz; α-synM: 11.7 ± 2.4 Hz; n = 4; p = 0.9162; Paired T-test). In summary, these data indicate that α-synM induced inhibitory membrane effects on neurons of LDTF, and a reduction in the amplitude and frequency of sEPSCs, which were opposite effects from those we have reported before in LDTM . In SNF, α-synM also induced inhibitory effects on the membrane, which were similar to those effects we have reported before in SNM. Further, α-synM had similar effects on synaptic events in SNM to those seen in SNF as in both sexes we observed a reduction in the amplitude of sEPSCs with no effect on frequency. Taken together, our findings show that the examined effects of α-synM on LDT neurons are sex-dependent, whereas, α-synM effects on SN are independent of sex.
Sex differences in LDT neurons of α-synM-mediated alteration of intracellular calcium
As α-synM-induced neuronal effects have been hypothesized to lead to calcium dysregulation [23, 28], we previously examined actions of this protein on intracellular calcium levels and found that α-synM induced changes in calcium in LDTM. We repeated those experiments here and confirmed our earlier findings. Using multiple-cell calcium imaging to monitor changes in Fura 2-AM fluorescence which were induced by α-synM (100 nM, 3 min), we observed changes in fluorescence in the majority of LDTM cells (97.4%; n = 38/39; Fig. 2B1), and the majority of responses were indicative of an increase in calcium (76.3%; n = 29/38; Fig. 2A1a, B2). We then examined responses in LDTF and found that α-synM induced changes in intracellular calcium in 100% (n = 89/89) of the examined cells (Fig. 2B1). However, interestingly, the majority of responding LDTF neurons exhibited changes in fluorescence indicative of a decrease in intracellular calcium levels (58.4%, n = 52/89; Fig. 2A2b, B2). When we compared alterations in intracellular calcium induced by α-synM in LDTM to LDTF, there was no significant difference in the numbers of responding or non-responding neurons (p = 0.3047; Fischer’s Exact test; Fig. 2B1). In contrast, there was a difference between males and females in the ratio of increases in calcium to decreases in calcium in response to α-synM. We observed a significantly higher proportion of cells responding with a decrease in calcium in LDTF when compared to the proportion of cells exhibiting decreases in LDTM (p = 0.0004; Fisher’s Exact test; Fig. 2B2).
Evaluation of the mechanism of α-syn M inhibition induced in the membrane of LDT neurons
To gain more information regarding the sex-based difference in the mechanism underlying α-synM membrane effects, we examined α-synM actions during blockade of the generation of Na+-dependent action potentials by inclusion in the bath of tetrodotoxin (TTX, 500 nM). Unexpectedly, in presence of TTX, α-synM induced an inward current (-7.7 ± 2.0 pA, n = 4), and this effect was present in all neurons tested (Fig. 3A, B).
Our findings with TTX indicated that α-synM-induced outward currents relied on a presynaptic mechanism. To confirm the involvement of a presynaptically-mediated mechanism in the induction of outward currents in the postsynaptic membrane of LDTF neurons, we next applied α-synM in a reduced calcium solution, which effectively eliminates calcium-dependent synaptic transmission. Because we wished to use a within cell control, we first verified whether a second application of α-synM to the same cell could result in similar effects on the membrane as those elicited in a first application. Thus, in a subset of LDTF neurons, we reapplied α-synM following a first application, and we observed that a second inhibitory response was elicited in all neurons tested, which did not significantly vary in amplitude from the outward current obtained in the first application (n = 3). Consistent with the TTX data, in all the cells tested in which the first application elicited an outward current, in the second application in presence of low calcium solution, we did not observe an induction of an outward current, and instead, an inward current was elicited (-16.1 ± 5.5 pA; n = 3; Fig. 3A2, B). Taken together, these data indicate that the inhibitory effect induced by α-synM on the membrane of LDTF involves presynaptic-dependent mechanisms.
LDT neurons receive a heavy inhibitory input from presynaptic GABAergic terminals both from local LDT neurons but also from projections sourcing from outside the nucleus , and, accordingly, GABAergic presynaptic mechanisms could be involved in α-synM-mediated inhibitory responses in LDTF. Therefore, we investigated α-synM-induced membrane responses in LDTF in presence of the GABA receptor antagonists, SR-95,531 (10 µM) and CGP-55,845 (10 µM), which block GABAA and GABAB receptors, respectively. Although no evidence has been presented of glycine-mediated inhibition of LDT cells, and we have not noted any glycinergically-mediated spontaneous inhibitory currents (sIPSCs) in our own studies under our recording conditions, we also included strychnine (2.5 µM) in the ACSF to ensure the blockade of any glycine-mediated events. In a population of cells in which the first application of α-synM induced an outward current, we found that in the presence of GABA and glycine receptor blockade, an inward current was elicited in all tested cells (-16.3 ± 5.2 pA; n = 3; Fig. 3A3, B1, 2).
When taken together, our data indicate that induction of outward current in LDTF neurons is reliant on inhibitory transmission from presynaptic neurons. Blockade of inhibitory transmission revealed an inward membrane current similar to what has been seen in LDTM. Although not tested in the present study, we showed in our previous work that inward currents in LDTM were mediated by a G-protein receptor coupled mechanism in postsynaptic neurons. Although we conducted experiments to examine a role for receptors previously implicated in α-synM effects, we could not identify the specific receptor involved; however, we speculate that this same excitatory mechanism is being activated in in LDTF but masked by the concurrent induction of outward current induced by α-synM-mediated stimulation of inhibitory presynaptic transmission.
Inhibitory amino acids are involved in the decrease of intracellular calcium
As we had seen that membrane current effects of α-synM involved inhibitory transmission, we examined whether similar mechanisms were also involved in the decrease of intracellular calcium seen in response to α-synM in the majority of neurons of the LDTF. During blockade of GABA and glycine receptors, while decreases in fluorescence indicative of reductions in calcium were still elicited, the amplitude of the decrease in fluorescence was significantly smaller (36%) compared to that elicited in control conditions (control: 57.1 ± 3.0% DF/F, n = 52; blockers: 36.2 ± 2.3% DF/F, n = 49; p = 0.0001; Paired T-test; Fig. 3C). Taken together, while they suggest that other mechanisms might be involved in the reductions in intracellular calcium induced by α-synM, these data provide evidence that inhibitory amino acids, most likely GABA contribute to α-synM-mediated calcium decreases in LDTF and provide further support that inhibitory transmission targeting postsynaptic LDT cells is activated by α-synM.
α-synM Reduces the excitability of LDT neurons in the female
We previously reported that α-synM enhanced the firing frequency of neurons within LDTM. However, the inhibitory effect induced by α-synM on the membrane of LDTF in conjunction with the reduction in amplitude and frequency of EPSCs could reduce neuronal excitability in the female. To directly investigate the functional effect of α-synM-mediated actions on LDTF neurons which could affect the output of these cells, we examined the firing frequency in current clamp mode following depolarization of the membrane of LDTF neurons sufficiently to induce action potentials (VM: -45.0 ± 5.0 mV) before and after application of α-synM. Under these conditions, α-synM reduced the firing frequency by 36.5% from baseline levels (control: 0.41 ± 0.05 Hz; α-synM: 0.26 ± 0.08 Hz; n = 3; p = 0.0498; Paired T-test; Fig. 4A, B). These data suggest that in direct contrast to findings in LDTM, functional actions of α-synM include reductions in neuronal excitability of neurons in LDTF, which would be expected to alter output of these cells to target regions.
α-syn M induces a lower cell death of LDT neurons in females compared to males
In our previous report, α-synM-mediated excitation of the membrane of neurons with a concurrent rise of intracellular calcium was suspected to induce excitotoxicity, which was supported by a heightened cell death over control in LDTM . As α-synM-induced inhibitory membrane current, and increases in calcium were less prominent in LDTF neurons, we hypothesized that neurodegeneration induced by α-synM would also exhibit a sex-based difference. First, we needed to determine whether α-synM induced cell death in LDTF above control. Accordingly, we evaluated cell survival in LDTF hemi slices in which one half had been exposed for 7 h to ACSF and the other half to 7 h in α-synM. We found a relatively lower cell survival in the half of the slice exposed to α-synM when normalized to survival seen in control (Cell Survival: Control: ACSF: 100 ± 0.9%, na = 217/n40 = 57; α-synM: 92.8 ± 1.5%, na = 183/n40 = 48; Fig. 5A).
Next, we compared cell survival in the halves of the LDTF exposed to α-synM for 7 h to halves of brain slices of the LDTM that had been similarly exposed to 7 h of α-synM. Supporting our hypothesis of a sex difference in α-synM cellular effects, we noted a significantly greater cell survival in the LDTF when compared to cell survival seen in the LDTM (Cell Survival: Female: 87.4 ± 0.6%, na = 183/n40 = 48; Males: 79.9 ± 0.6%, na = 168/n40 = 66, p < 0.0001; Unpaired Student’s T-test; Fig. 5A). These results indicate that α-synM induces toxic effects in neurons of the LDTF to a lesser degree than in LDTM.
Endogenous neuroprotection against α-synM induced degeneration in LDT neurons from female involved inhibitory transmission
As we showed a sex-based differential effect of α-synM on neuronal mortality in the LDT which was reminiscent of the sex-based difference in membrane excitability and changes in intracellular calcium levels induced by this protein which were affected by GABA and glycine receptor antagonists, we reasoned that the differential effect on neurodegeneration could involve inhibitory amino acid activity which was protective in the LDTF. To examine this hypothesis, we exposed LDTF cells to α-synM for 7 h in the presence of antagonists of GABAA, GABAB and glycine receptors (G-ANT) and normalized cell viability to that in the other halves of the bisected slices that were exposed only to α-synM. The relative degree of LDTF of cell survival associated with α-synM in the presence of G-ANT was significantly lower than in absence of blockers of receptors of inhibitory amino acids (Cell survival: α-synM: 100.0 ± 2.4%, na = 199/n40 = 44, α-synM + G-ANT: 88.8 ± 1.5%, na = 169/n40 = 35; p = 0.0001; Mann-Whitney Test; Fig. 5B). These results indicate that the neuroprotective effects seen in the female brain against α-synM-mediated toxicity involve functional inhibitory amino acid transmission.
Activation of GABAergic transmission in male brain induces neuroprotection against α-synM -induced neurodegeneration
Next, we hypothesized that activation of GABA receptors could be neuroprotective against α-synM-mediated toxicity in LDTM. To examine this hypothesis, we exposed LDTM neurons to α-synM for 7 h in the presence of the GABAA, and GABAB agonists, muscimol and baclofen (G-AGO), and cell death was compared to that in the other halves of the bisected slices exposed only to α-synM. Remarkably, when exposed to α-synM the degree of cell survival seen in the halves of the slice treated with GABA receptor agonists was significantly higher than that in the other halves of the bisected slices exposed only to α-synM (Cell Survival: ⍺-synM: 100.0 ± 2.3%, na = 168/n40 = 66, α-synM + G-AGO; 106.0 ± 2.7%, na = 127 /n40 = 48, p = 0.0334; Mann-Whitney Test; Fig. 5C). These results provide further support for the conclusion that in LDTF, a GABAergic-mediated mechanism protects against α-synM-induced toxicity, and, excitingly, indicate that activation of GABA mechanisms could protect neurons of the LDTM from α-synM-mediated neurodegeneration.
We found that in contrast to what we previously saw in LDTM, effects of α-synM on the membrane of LDTF neurons were inhibitory, and we recorded decreases in excitatory synaptic events, reductions in firing rate, and relatively more decreases in intracellular calcium. Further, cell death associated with α-synM was lower in females than in males. Changes in membrane currents and synaptic excitability noted in the SN did not exhibit a sex-based difference, suggesting nucleus specificity of α-synM-mediated effects. Inhibitory membrane currents and reductions in calcium induced in LDTF were found to involve inhibitory transmission, which when blocked revealed membrane excitation similar to that seen in LDTM. Finally, consistent with a protective role of inhibitory signaling, blockade of GABAA, GABAB, and glycine neurotransmission in the LDT of the female resulted in greater cell death and activation of GABAergic receptors reduced α-synM-mediated neurodegeneration in the LDT of the male.
While the polarity of α-synM-induced membrane effects on LDTF neurons was opposite from that seen in our earlier study conducted in LDTM, when presynaptic input was blocked, an excitatory membrane response similar to that seen in LDTM neurons was revealed. This leads us to the interpretation that α-synM induces a dual effect on the membrane of neurons of the LDTF with the summation resulting in inhibition of membrane currents of the postsynaptic neuron. The mechanism underlying the occlusion of excitatory membrane actions putatively involved GABA-releasing, presynaptic neurons, although glycinergic mechanisms were not ruled out. Sex-based differences were also found in the polarity of calcium responses between male and female in that a decrease of intracellular calcium was observed in the majority of neurons of the LDTF, whereas in LDTM, the majority of responses were indicative of rises in intracellular calcium. Similar to the membrane responses, the decrease in intracellular calcium induced by α-synM in female involved inhibitory amino acids, suggesting sex-based differences in inhibitory transmission in the LDT. Cell death associated with α-synM was lower in females but increased when GABA was blocked suggesting that GABA is involved in inhibiting neurodegenerative processes. Consistent with this, the presence of GABA receptor acting agents was able to prevent neurodegeneration in the male LDT.
Besides suggesting potential targets to inhibit neurodegeneration, our data suggest presence of a GABAergic system in the female LDT, which leads to reductions in excitatory cellular effects and cell death and that this system does not function similarly in the male LDT. While not examined specifically within the LDT, the GABAergic system has shown sexual dimorphism in the brain. In both young and adult mice, expression of proteins involved in GABA synthesis and metabolism, as well as presence of GABAA receptors have shown sex-based differences [30,31,32,33,34,35]. Further, the numbers of GABAergic neurons, as well as the responsiveness to GABA-acting drugs, have been shown to be associated with sex [36, 37]. The sex-specific phenotypic and functional differences in the GABAergic system may play key roles in the differential sensitivity of the LDTM and LDTF to α-synM [31, 34, 38, 39].
Our study has several limitations. Patch clamp recordings and multiple-cell calcium imaging with Fura-AM is difficult in slices from old animals, and thus it remains unknown if the sex-dependent effect of α-synM continues across ontogeny, which is relevant to neurodegeneration which is expected to increase across age. Further, we did not identify the phenotype of cells that were protected in presence of GABAA, and GABAB receptor agonists. Since loss of cholinergic cells in the LDT and the neighboring pedunculopontine tegmentum has been one neuropathological feature noted in α-syn-related diseases [40, 41], we tried to target large neurons with the cholinergic profile , however, while we do not believe we recorded from many, if any, GABA cells as they are much smaller [17, 24], non-cholinergic neurons could have been included. Nevertheless, while we did not identify the LDTF cell phenotypes exhibiting inhibitory membrane responses to α-synM, we did show in our earlier work that excitatory cellular responses in the LDTM were transmitter phenotype-independent . Finally, while we did examine a nucleus which was central in SDs, given the global nature of sleep, it is almost certain that networks of nuclei, and not just activity in one nucleus mediate aberrant sleep behaviors seen in neurodegenerative diseases. Accordingly, future studies should examine effects across a larger age span, identify cellular phenotype, and conduct recordings across multiple sleep-controlling nuclei.
Despite the limitations, our work is based on multiple strengths, which differ from other investigations. Most studies of cellular effects of α-syn have been conducted using the oligomeric form of α-syn, and thus our data provides important information about effects of the monomeric form. Further, while the focus of much work has been on targeting α-syn intracellular exposure, we have utilized extracellular exposure. Additionally, in many studies, concentrations applied have been higher than those seen during pathological conditions (from 0.5 µM to 5 µM) ; however, we have used nanomolar concentrations of highly purified α-synM, which more accurately reflects the clinical condition. Moreover, few studies have used ex vivo brain tissue but rather cultured cells; thus, our findings more directly add to the body of knowledge of effects in native mammalian tissue. Finally, to the best of our knowledge, no one has previously reported a sex-based difference in cellular effects of α-synM on any neuronal type in ex vivo studies. α-synM was shown to induce an inhibitory effect in synaptic transmission in Calyx of Held; however, while both male and female rats were used, data were not analyzed for potential sex-based differences . Taken together, this constitutes the first report to show that α-synM at a concentration reflective of clinical exposures induces a sex-dependent, cellular effect in mammalian neurons. Furthermore, as no difference in membrane effects was observed between LDTF and SNF, but we did see differences between the LDTM and SNM in earlier work , this suggests that α-synM induces sex-dependent effects in specific brain nuclei. Future studies of α-synM effects should consider sex as a factor as well as regional differences.
The observed sex-based, different cellular responses likely have pervasive functional implications for behaviors and symptoms when neurons of the LDT are exposed to α-synM. We have a working hypothesis that prodromal SDs in PD could be due to early dysfunction of sleep controlling nuclei, including the LDT, which has been implicated in RBD and EDS [23, 42, 44, 45].
Central to this hypothesis, we suspect that as levels of the monomeric form of α-syn rise, cellular effects are exerted on LDT neurons, which include enhancement of cellular excitability and increases in levels of intracellular calcium. Such effects are not elicited in the SN by the monomeric form, and it is believed to be the later appearing forms of aggregated oligomeric and fibril α-syn which produces neurodegeneration in the SN [23, 46,47,48,49,50]. Sustained elevation of excitability and calcium levels in the LDT induced by α-synM could trigger neurodegenerative processes when cells are excited for extended periods, and when calcium levels remain high [51, 52]. Consistent with this, we have shown that the effects induced by α-synM were associated with neuronal death in LDTM neurons. However, in SNM neurons in which α-synM induced an inhibitory membrane effect, and the predominant response was a decrease in intracellular calcium, no differences in neuronal survival were noted, which shows that α-synM cellular actions do not necessarily lead to degeneration as we saw in the LDT .
Interestingly, in the LDTF, α-synM induced very similar effects on the membrane and synaptic events to those seen in the SNM leading us to suggest that these effects are neuroprotective in the female LDT. This tenant is supported by greater cell viability in the LDT of the female compared to that in the male following α-synM exposure. The neuroprotective mechanisms involved inhibitory amino acid transmission as the blockade of GABA and glycine receptors revealed an excitatory effect in the LDTF similar to that seen in LDTM, which we hypothesize could underlie cellular degeneration. The outward current was sufficient to mask the concurrent excitatory effect and presumably limit putative damage from α-synM-mediated excitation. Further, treatment with GABA receptor agonists resulted in reductions in cell death in the LDTM lending further support to the interpretation that GABA signaling is neuroprotective. We did not identify the source of the putative, protective GABAergic effect in LDTF; however, elucidation of the source of this GABA tone in LDTF is of great interest and will be a focus of future studies. Also likely contributing to α-synM-induced neurodegeneration in the male LDT were the sex-dependent differences in the firing frequency, as a reduction in firing was induced in the LDTF by α-synM, whereas an enhancement in firing was seen in our earlier study in the LDTM . Over the long-term, increases of neuronal discharge can produce overexcitability-induced cell death since high-levels of excitability and firing elevates glutamate exposure, which results in alterations of intracellular calcium levels. This can trigger apoptotic events and collapse of mitochondrial functions which are all processes contributing to neuronal death [53,54,55,56,57,58]. Accordingly, the α-synM-induced reduction in neuronal firing seen in LDTF could exert a protective effect.
One implication of our findings is that processes controlled by the LDT that could be affected in PD are less likely to be affected in females. While speculative, our data do support clinical findings related to occurrence of LDT-involved sleeping disorders seen prodromal to PD. RBD and EDS appear to be more common in PD males as well as in males during the prodromal phase. The majority of patients diagnosed with RBD are male, with the reported percentage of females in these studies ranging from 10 to 17.5% of all diagnosed cases [9, 10, 45, 59,60,61,62]. Although very few studies have focused on differences in SD symptoms between men and women, sex differences in RBD symptomatology have been reported with aggressive and violent motor active RBD behaviors appearing more commonly in men than in women [63, 64]. EDS is characterized by the incapacity of the individual to stay awake during the circadian day due to excessive sleepiness. Several studies have examined the risk of development of PD in EDS patients; however, the majority of investigations which have shown an association between EDS in the prodromal phase of PD have been conducted in males [13, 65]. In one of the few studies to include both sexes, a higher risk of development of PD was documented in those expressing sleepiness during the day; however, the data were not analyzed to compare the risk in males vs. females, and the majority of the cohort who exhibited EDS were males, which reflects the sex-based odds ratio of PD in the general population . In several studies of PD diagnosed patients, EDS has been shown to be more common among PD-affected men than women [65, 67, 68].
Taken together, our data lead us to conclude that the cellular effects exerted by α-synM are neuroprotective in the LDTF and could be sufficient to delay α-synM-mediated cell death in this nucleus, perhaps ceasing when the relevant GABAergic neurons perish as neuronal loss proceeds throughout the PD affected brain. Output from the LDT to rostral and caudal targets is implicated in control of arousal during wakefulness, governance of the sleep and wakefulness cycle, and maintenance of motor atonia, which is a hallmark of REM sleep [18, 69]. Thus, if GABAergic neurotransmission plays a neuroprotective role by leading to outward currents and reductions in intracellular calcium, thereby counterbalancing negative effects induced by PD processes, this could block loss of cells in the LDT that produce motor atonia during REM sleep and an aroused EEG during wakefulness and sleep, and thereby lead to the lower frequency of RBD and EDS symptoms see in female when compared to those seen in male patients with PD [10, 70, 71].
As we observed no sex-based difference in cellular responses in the SN, our findings cannot account for the higher incidence of PD in males compared to females; however, we do suggest a mechanistic basis for the higher prevalence of SDs among male vs. female PD patients. Thus, our findings represent an important step toward the identification of sex differences in the mechanisms underlying the pathology of α-syn-associated neurogenerative diseases. Such identification offers the potential of targeting inhibitory mechanisms as neuroprotective strategies in neurodegenerative diseases, and to speed efforts for development of new directions for PD treatment and management in the prodromal phase of these diseases.
All data are available upon reasonable request made to the corresponding author.
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Christel Ammitzböll Halberg is acknowledged for technical assistance.
Open access funding provided by Royal Library, Copenhagen University Library. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil is acknowledged for funding support in the form of a Ph.D Grant to Altair Brito dos Santos. AEL acknowledges funding from the Lundbeck Foundation Initiative BRAINSTRUC (2015–2666).
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Santos, A.B.D., Thaneshwaran, S., Ali, L.K. et al. Sex-dependent neuronal effects of α-synuclein reveal that GABAergic transmission is neuroprotective of sleep-controlling neurons. Cell Biosci 13, 172 (2023). https://doi.org/10.1186/s13578-023-01105-4