# Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease

Sabina Luchetti, Philippe Liere, Antoine Pianos, Ronald W H Verwer, Arja

Sluiter, Inge Huitinga, Michael Schumacher, Dick F Swaab, Matthew R J

Mason

► To cite this version:

Sabina Luchetti, Philippe Liere, Antoine Pianos, Ronald W H Verwer, Arja Sluiter, et al. Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease. Neurobiology of Disease, 2023, 183, pp.106169. 10.1016/j.nbd.2023.106169. inserm-05354168

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# Disease stage-dependent changes in brain levels and neuroprotective effects of neuroactive steroids in Parkinson's disease

Sabina Luchetti<sup>a,b</sup>, Philippe Liere<sup>c</sup>, Antoine Pianos<sup>c</sup>, Ronald W.H. Verwer<sup>a</sup>, Arja Sluiter<sup>a</sup>, Inge Huitinga<sup>b</sup>, Michael Schumacher<sup>c</sup>, Dick F. Swaab<sup>a</sup>, Matthew R.J. Mason<sup>b,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Neurosteroids

GC/MS

Allopregnanolone

5α-dihydroprogesterone

Slice culture

Post-mortem

## ABSTRACT

Neuroactive steroids are known neuroprotective agents and neurotransmitter regulators. We previously found that expression of the enzymes synthesizing 5α-dihydroprogesterone (5α-DHP), allopregnanolone (ALLO), and dehydroepiandrosterone sulfate (DHEAS) were reduced in the substantia nigra (SN) of Parkinson's Disease (PD) brain. Here, concentrations of a comprehensive panel of steroids were measured in human post-mortem brains of PD patients and controls. Gas chromatography-mass spectrometry (GC/MS) was used to measure steroid levels in SN (involved in early symptoms) and prefrontal cortex (PFC) (involved later in the disease) of five control (CTR) and nine PD donors, divided into two groups: PD4 (PD-Braak stages 1–4) and PD6 (PD-Braak stages 5–6).

In SN, ALLO was increased in PD4 compared to CTR and 5α-DHP and ALLO levels were diminished in PD6 compared to PD4. The ALLO metabolite 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was higher in PD4 compared to CTR. In PFC, 3α5α20α-HHP was higher in PD4 compared to both CTR and PD6.

The effects of 5α-DHP, ALLO and DHEAS were tested on human post-mortem brain slices of patients and controls in culture. RNA expression of genes involved in neuroprotection, neuroinflammation and neurotransmission was analysed after 5 days of incubation with each steroid. In PD6 slices, both 5α-DHP and ALLO induced an increase of the glutamate reuptake effector GLAST1, while 5α-DHP also increased gene expression of the neuroprotective TGFB. In CTR slices, ALLO caused reduced expression of IGF1 and GLS, while DHEAS reduced the expression of p75 and the anti-apoptotic molecule APAF1.

Together these data suggest that a potentially protective upregulation of ALLO occurs at early stages of PD, followed by a downregulation of progesterone metabolites at later stages that may exacerbate the pathological changes, especially in SN. Neuroprotective effects of neurosteroids are thus dependent on the neuropathological stage of the disease.

## 1. Introduction

The underlying causes of non-familial Parkinson's disease (PD) are poorly understood and there is an outstanding need for therapeutic agents which will combat the symptoms and prevent or slow down the ongoing neurodegeneration. Neuroactive steroids regulate important neural functions and represent a novel class of target molecules that may fulfil a therapeutic role in PD (Brinton, 2013; Guennoun et al., 2015; Schumacher et al., 2007a, 2007b). Neuroactive steroids can be synthesized in steroidogenic glands and accumulated/metabolized in the brain (e.g. steroid sulfates, progestogens, glucocorticoids, androgens and

estrogens), but can also be locally produced in neurons and glial cells (neurosteroids) from cholesterol and/or from circulating precursors (Baulieu et al., 2001). Local synthesis of neuroactive steroids, such as progesterone and its 5α-reduced metabolites 5α-dihydroprogesterone (5α-DHP) and allopregnanolone (ALLO) may be of particular importance for regulating steroidal effects on the nervous system (Luchetti et al., 2011).

ALLO is known to be a highly potent positive allosteric modulator of GABA-A receptors (Belelli and Lambert, 2005; Carver and Reddy, 2013), increasing inhibitory neurotransmission and so providing neuroprotection by counteracting excitotoxicity. ALLO is also a potent ligand

<footnote><sup>a</sup> Neuropsychiatric Disorders Group, Netherlands Institute for Neuroscience (NIN), Meibergdreef 47, 1105, BA, Amsterdam, the Netherlands</footnote>

<footnote><sup>b</sup> Neuroimmunology Research Group, NIN, Amsterdam, the Netherlands</footnote>

<footnote><sup>c</sup> U1195 INSERM and University Paris Saclay, Le Kremlin Bicêtre, 94276 Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: m.mason@nin.knaw.nl (M.R.J. Mason).</footnote>
of membrane progesterone receptors (mPRs) belonging to the PAQR family and exerts anti-apoptotic effects via these receptors on neural cells (Thomas and Pang, 2020). Progesterone and 5α-DHP act via the nuclear Progesterone Receptor (PR), which acts as a transcription factor to promote expression of target genes. Progesterone has neuroprotective activity, partly via conversion to ALLO, but also directly via the mPRs, and via the nuclear PR (Guennoun et al., 2015).

Neuroactive steroids, therefore, are endogenous neuroprotectants. Many animal studies have shown that these steroids have beneficial effects in the aging brain, in traumatic or ischemic CNS injury models, and during motoneuron degeneration and can stimulate myelination by oligodendrocytes (Bassani et al., 2022; Schumacher et al., 2014). ALLO and its precursor 5α-DHP have been shown to protect neurons against NMDA and kainic acid-induced excitotoxicity (Ciriza et al., 2004; Lockhart et al., 2002), and decrease apoptosis (Djebaili et al., 2005, 2004; He et al., 2004). Neuroactive steroids have beneficial effects in animal models of PD but their clinical potential is not clear (Bourque and Di Paolo, 2022). In particular, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, progesterone protected dopaminergic neurons from degeneration and preserved striatal dopamine (Bourque et al., 2016, 2009). Furthermore, progesterone was found to increase dopamine release and the gene expression of dopamine transporter in the striatum (Sanchez et al., 2010). Thus, progesterone and its metabolite ALLO can influence the GABAergic and dopaminergic systems which are profoundly affected in PD.

Therefore, neuroactive steroids may act on multiple pathological aspects of PD. In human brain studies, Bixo et al. (Bixo et al., 1997) found relatively high levels of progesterone, 5α-DHP and ALLO in the substantia nigra (SN) and striatum of female control subjects, indicating that synthesis and accumulation of these neuroactive steroids take place in the nigrostriatal dopaminergic areas where neurodegeneration occurs in PD. We found initial evidence that neurosteroid levels were impaired in PD. Levels of 5α-DHP and ALLO were reduced in the cerebrospinal fluid (CSF) and plasma of PD patients (di Michele et al., 2003), and we also observed a decrease in gene expression of 5α-reductase type 1 (5α-R1), the enzyme that synthesizes 5α-DHP, the direct precursor of ALLO, in peripheral mononuclear blood cells (Luchetti et al., 2006). Previously, we identified the steroid synthetic enzymes as a novel class of genes that are altered in PD in the SN. The mRNA expression levels of 5α-R1 and SULT2B1, which synthesizes dehydroepiandrosterone-sulfate (DHEAS) from DHEA, were decreased in neurons of SN in PD patients (Luchetti et al., 2010). Since DHEAS and ALLO are neuroprotective agents, a downregulation of their synthesis might exacerbate the neurodegenerative process (Luchetti et al., 2010; Maninger et al., 2009). Moreover, since synthesis alterations of neuroactive steroids were observed in neurons that have not yet degenerated (Bossers et al., 2009) this suggests an early, possibly causative role in PD.

Here we analysed a panel of steroids by gas chromatography coupled to mass spectrometry (GC/MS) in brain tissue of PD patients and control donors. Steroids were quantified in SN (which degenerates earlier in PD) and prefrontal cortex (PFC) (which degenerates later in the disease) at two PD neuropathological stages to detect changes that might be important for neuroprotection/neurodegeneration. In addition, because the gene expression changes we found previously (Luchetti et al., 2010) suggested reduced synthesis of 5α-DHP, ALLO and DHEAS, we analysed the effects on gene expression of treatment with these three steroids on human brain slice cultures of control and PD donors. This was done using a unique human post-mortem organotypic slice culture assay which was previously developed in our laboratory (Verwer et al., 2002a, b). Since PFC is involved late in the neurodegenerative process of PD (Braak et al., 2003), we chose this area to look into possible effects of steroid treatment, so that potential protective effects may be more apparent. Gene expression of neurotrophic factors, pro-apoptotic molecules, PD risk-associated genes, anti-inflammatory molecules, genes involved with GABA synthesis or glutamate synthesis and reuptake, cell-type markers, and molecular chaperones were analysed by qPCR.

## 2. Material and methods

### 2.1. Post-mortem material

Post-mortem brain samples were obtained through the rapid autopsy program of Netherlands Brain Bank (NBB) and systematically neuro-pathologically investigated. The NBB obtained permission from its donors or next of kin for brain autopsy and the use of the brain samples and clinical data for research purposes.

The autopsy, use of tissue and clinical information for research purposes by the NBB was approved by the Ethical Committee of the Medical Faculty of the Free University (VUMC, Amsterdam, The Netherlands, 971,019) where the autopsies were performed.

Subjects were matched for age, sex, post-mortem interval and CSF pH as closely as possible. In addition, we included only patients who were not treated with corticosteroids, indometacin, benzodiazepine or hormonal therapies in the last 3 months before death.

Based on the neuropathological stage of PD, relative to the brain distribution of Lewy body (aberrant α-synuclein protein aggregations) (Braak et al., 2003), PD patients were divided in two groups followed by Braak stage number (in order to distinguish from Alzheimer stages) as: PD4 (includes Braak 1-4 from preclinical to initial symptoms) and PD6 (includes Braak 5-6 with advanced disease). For the GC/MS experiments, frozen samples of PFC and SN of PD4 (n = 4), PD6 (n = 5) and control subject (CTR, n = 5) without a neurological or psychiatric disease were used. Clinico-pathological information of the patients are shown in Table 1. No significant differences were found in age, post-mortem delay, CSF pH or brain weight between the three groups.

For the slice culture experiments, fresh brain tissue from the PFC of PD4 (n = 4), PD6 (n = 5) and CTRs (n = 6) was used (see Table 1 for clinico-pathological information). No significant differences were found in post-mortem delay, CSF pH or brain weight between the three groups.

### 2.2. Steroid profiling by gas chromatography-mass spectrometry (GC/MS)

Steroids measurements were performed by GC/MS according to the protocol described elsewhere (Gonzalez Deniselle et al., 2016; Hertig et al., 2010) with minor modifications. Steroids were extracted with 10 volumes of methanol from 50 to 150 mg of SN and from aliquots of 400 mg of PFC. The following internal standards were added to the extracts for steroid quantification: 2 ng of $ ^{2}H_{4} $-pregnenolone sulfate (for PREGS), 5 ng de $ ^{2}H_{6} $-DHEAS (for DHEAS), 2 ng of $ ^{2}H_{8} $-5α-dihydroprogesterone (for 5α-DHP and 5α-DHADIONE), 2 ng of 19-nor progesterone (for progesterone, 20α-DHP, 3α/β5α/β-THP, 3α5α20α-HHP, 3α5α-THDOC), 2 ng of epietiocholanolone (for PREG, 20α-DHPREG, 5α-DHT, 3α5α-THT, DHEA, etiocholanolone, epiandrosterone), 2 ng of $ ^{2}H_{5} $-testosterone (for testosterone), 2 ng of $ ^{2}H_{7} $-androstenedione (for androstenedione), 2 ng of $ ^{2}H_{5} $-17β-E2 (for 17β-E2 and E1), 2 ng of $ ^{2}H_{8} $-deoxycorticosterone (for DOC), 10 ng of $ ^{2}H_{8} $-corticosterone (for corticosterone), 50 ng $ ^{2}H_{4} $-cortisol (for cortisol and 11-deoxycortisol), and 50 ng $ ^{2}H_{7} $-cortisone (for cortisone). Samples were purified and fractionated by solid-phase extraction with the recycling procedure (Liere et al., 2004) and by high performance liquid chromatography. Samples were derivatized and analysed by GC/MS as described elsewhere using an AS 3000 autosampler (Thermoscientific, USA), a Focus gas chromatograph (GC) coupled with a DSQII mass spectrometer (Thermoscientific) (Gonzalez Deniselle et al., 2016).

The analytical protocol has been previously validated for all the screened steroids by using a pool of brain of male rats. The calculations of the limit of detection, linearity, accuracy and inter-assay precision have shown that the used GC/MS methodology is suitable for steroids measurements in brain (Meffre et al., 2007). The steroids analysed with their abbreviations used in the text are listed in Table 2.
<table><caption>**Table 1**
Clinico-pathological information of control donors and PD patients.</caption><thead><tr><th>Experiment</th><th>Group</th><th>Sex</th><th>Age</th><th>Braak LB</th><th>PMD</th><th>pH</th><th>BW (g)</th><th>Cause of death</th></tr></thead><tbody><tr><td rowspan="16">GC/MS</td><td>CTR</td><td>f</td><td>89</td><td>0</td><td>03:52</td><td>7.3</td><td>1268</td><td>pneumonia</td></tr><tr><td>CTR</td><td>f</td><td>85</td><td>0</td><td>05:20</td><td>7.07</td><td>1176</td><td>natural death</td></tr><tr><td>CTR</td><td>m</td><td>82</td><td>0</td><td>05:10</td><td>6.75</td><td>1132</td><td>respiratory failure</td></tr><tr><td>CTR</td><td>m</td><td>83</td><td>0</td><td>05:45</td><td>6.35</td><td>1590</td><td>pneumonia</td></tr><tr><td>CTR</td><td>m</td><td>76</td><td>0</td><td>06:45</td><td>6.31</td><td>1220</td><td>lung cancer</td></tr><tr><td>PD4</td><td>f</td><td>83</td><td>4</td><td>06:05</td><td>6.32</td><td>1075</td><td>cachexia and dehydration</td></tr><tr><td>PD4</td><td>f</td><td>80</td><td>4</td><td>06:50</td><td>6.5</td><td>1265</td><td>respiratory and heart failure</td></tr><tr><td>PD4</td><td>m</td><td>92</td><td>3</td><td>07:00</td><td>6.76</td><td>1187</td><td>heart failure</td></tr><tr><td>PD4</td><td>m</td><td>78</td><td>4</td><td>06:15</td><td>6.33</td><td></td><td>pneumonia</td></tr><tr><td>PD6</td><td>f</td><td>88</td><td>6</td><td>04:15</td><td>6.56</td><td>1190</td><td>legal euthanasia</td></tr><tr><td>PD6</td><td>m</td><td>84</td><td>6</td><td>06:48</td><td>6</td><td>1249</td><td>dehydration/infection</td></tr><tr><td>PD6</td><td>m</td><td>81</td><td>6</td><td>05:15</td><td>6.78</td><td>1416</td><td>decompensation cordis</td></tr><tr><td>PD6</td><td>m</td><td>82</td><td>6</td><td>06:05</td><td>6.09</td><td>1092</td><td>choking asphyxia and dehydration</td></tr><tr><td>PD6</td><td>m</td><td>81</td><td>6</td><td>04:30</td><td>6.42</td><td>1315</td><td>legal euthanasia</td></tr><tr><td rowspan="16">Slice culture</td><td>CTR</td><td>f</td><td>89</td><td>0</td><td>06:35</td><td>6.7</td><td>1139</td><td>heart failure and dehydration</td></tr><tr><td>CTR</td><td>f</td><td>92</td><td>0</td><td>04:40</td><td>6.7</td><td>1206</td><td>respiratory failure</td></tr><tr><td>CTR</td><td>f</td><td>80</td><td>0</td><td>07:04</td><td>6.2</td><td>1450</td><td>legal euthanasia</td></tr><tr><td>CTR</td><td>m</td><td>90</td><td>0</td><td>05:45</td><td>6.4</td><td>1215</td><td>unknown</td></tr><tr><td>CTR</td><td>m</td><td>80</td><td>0</td><td>04:25</td><td>6.6</td><td>1429</td><td>legal euthanasia</td></tr><tr><td>CTR</td><td>m</td><td>85</td><td>0</td><td>07:25</td><td>6.5</td><td>1174</td><td>pneumonia</td></tr><tr><td>PD4</td><td>f</td><td>62</td><td>4</td><td>08:00</td><td>na</td><td>1400</td><td>legal euthanasia</td></tr><tr><td>PD4</td><td>f</td><td>85</td><td>4</td><td>06:40</td><td>6.8</td><td>1085</td><td>pneumonia</td></tr><tr><td>PD4</td><td>m</td><td>86</td><td>4</td><td>07:25</td><td>6.3</td><td>1113</td><td>cardiac arrest & respiratory tract infection</td></tr><tr><td>PD4</td><td>m</td><td>70</td><td>3</td><td>06:20</td><td>6.6</td><td>1155</td><td>pneumonia with cardiogenic shock</td></tr><tr><td>PD6</td><td>f</td><td>74</td><td>6</td><td>05:10</td><td>6.4</td><td>1313</td><td>legal euthanasia</td></tr><tr><td>PD6</td><td>m</td><td>67</td><td>6</td><td>06:35</td><td>na</td><td>1314</td><td>cachexia and dehydration</td></tr><tr><td>PD6</td><td>m</td><td>70</td><td>6</td><td>05:45</td><td>6.5</td><td>1303</td><td>unknown</td></tr><tr><td>PD6</td><td>m</td><td>74</td><td>6</td><td>04:35</td><td>6.6</td><td>1306</td><td>respiratory insufficiency and dehydration</td></tr><tr><td>PD6</td><td>m</td><td>87</td><td>6</td><td>04:45</td><td>6.4</td><td>1268</td><td>dehydration and infection</td></tr></tbody></table>

Abbreviations: Braak LB = Braak neuropathological stage for Lewy body; PMD = post mortem delay (hours:minutes); BW (g) = brain weight (grams); GC/MS = gas chromatography-mass spectrometry; CTR = control subjects; PD4 = Parkinson's disease subject at neuropathological Braak LB stage 1-4; PD6 = Parkinson's disease subject at neuropathological Braak LB stage 5-6; f = female; m = male.

## 2.3. Post-mortem brain slice culture

Brain tissue processing and culture procedures have been described in detail before (Qi et al., 2018; Verwer et al., 2002a; Verwer et al., 2002b; Wu et al., 2008). Briefly, PFC tissue was cut into 300-µm-thick slices comprising all cortical layers and a part of the white matter were obtained using a McIlwain tissue chopper. Slices were systematically distributed over 24-well plates and cultured free-floating. Each well contained one slice of tissue with 500 µL of Neurobasal A medium (Invitrogen) supplemented with gentamicin 0.1%, HEPES 0.5%, glutamax 1% and B27 2% (Qi et al., 2018). Slices were individually incubated with 5α-DHP, ALLO or DHEAS. 5α-DHP and ALLO were purchased from ACRIS Germany. DHEAS was purchased from Sigma Aldrich (cat. n. 700086P). For all the culture experiments, steroids were dissolved in ethanol which was used at a final concentration of 0.1%. Medium with ethanol at this concentration was also used for vehicle control incubations.

Steroids were added immediately at four different concentrations: 0.1 nM, 1 nM, 10 nM (low physiological brain concentrations) and 100 nM (a high physiological brain concentration) (Ahboucha et al., 2012; Bixo et al., 1997; Weill-Engerer et al., 2002). After two days in culture, 100ul medium plus steroids was refreshed. After 5 days in culture, tissue was collected, quickly frozen in liquid Nitrogen and stored at -80 °C until use.

In the first experiments, viability of the cells in the slices after steroid treatment was assessed using a Live/Dead kit (L3224, Life Technologies, Bleiswijk, the Netherlands) according to the manufacturer's protocol as previously done (Verwer et al., 2002b; Verwer et al., 2003).

## 2.4. Quantitative reverse transcriptase PCR (qPCR)

RNA expression levels of several human genes were determined using reverse transcriptase qPCR (RT-qPCR) as previously described

(Luchetti et al., 2010). Isolation of total RNA was performed using Tri-sure (Invitrogen). Complementary DNA was synthesized using a Quan-titec reverse transcription kit (cat. no. 205311; Qiagen, Benelux, Belgium). Gene expression was quantified for the following categories of genes: neurotrophic factors and signalling (BDNF, GDNF, NGF, NT3, TRKB); pro-apoptotic molecules (cJUN, APAF1, P75); PD risk (SNCA, APOE); anti-inflammatory molecules cytokines and growth factors (IGF1, IL10, IL4, TGFB, TNFA); GABA synthesis (GAD1, GAD2).

Glutamate synthesis and reuptake (GLS, GLAST, GLT1, GLUL); cell-type markers (AIF1, GFAP, HLA, MBP, OLIG2, RBFOX3, S100B, VIM); molecular chaperones (ABCRYST, HSP70, HSP90). The primer pairs and amplicon lengths are given in Supplementary Table 1.

For each RNA isolation, four slices per condition were used. Amplification was detected with ABI 7300 real-time qPCR systems using SYBR green master mix (Life Technologies). Controls omitting reverse transcriptase or template were included in each PCR reaction. qPCR was performed as previously described (Luchetti et al., 2010).

Primer and gene expression were normalized with the mRNA expression level of GAPDH as a reference gene, which was selected based on expression stability among a group of putative reference genes (Verwer et al., 2015).

## 2.5. Statistical analysis

Clinico-pathological information (age, post-mortem delay, CSF pH and brain weight values) was compared between groups with one-way ANOVA.

GC/MS data were analysed with one-way ANOVA. Some steroids displayed unequal variance across groups (heteroscedasticity), and were analysed using one-way ANOVA with the White-Huber correction. The p-values for the F-test were corrected for multiple comparisons using the Benjamini-Hochberg method. When significant, post-hoc comparisons were carried out using the Tukey post-hoc test. Outlier values
<caption>**Table 2**</caption>

Steroid measurements by GC/MS in SN and PFC from CTR and PD groups. Steroids are grouped by chemical structure family. Levels of steroids are expressed in nanogram/gram of tissue (ng/g) as median (Interquartile Range). CTR: Control donors (n = 5); PD4: PD patients in early neuropathological stages (Braak-LB stages 3–4, n = 4); PD6: PD patients in late neuropathological stages (Braak-LB stages 5–6, n = 5). SN: substantia nigra; PFC: pre-frontal cortex. -: not detected.

<table><thead><tr><th>Class of steroid</th><th>Steroid name</th><th>Abbreviation</th><th>CTR PFC</th><th>CTR SN</th><th>PD4 PFC</th><th>PD4 SN</th><th>PD6 PFC</th><th>PD6 SN</th></tr><tr><td></td><td></td><td></td><td colspan="6">ng/g</td></tr></thead><tbody><tr><td rowspan="5">Pregnenolone and<br>metabolites</td><td>Pregnenolone</td><td>PREG</td><td>18.9 (6.4)</td><td>14.8 (2.6)</td><td>24.3 (5.0)</td><td>18.0 (4.8)</td><td>4.7 (2.2)</td><td>5.0 (1.3)</td></tr><tr><td>20α-dihydropregnenolone</td><td>20α-DHPREG</td><td>0.67<br>(0.075)</td><td>2.6 (0.29)</td><td>0.59 (0.15)</td><td>1.2 (0.36)</td><td>0.47 (0.24)</td><td>1.7 (0.27)</td></tr><tr><td>Pregnenolone sulfate</td><td>PREGS</td><td>0.79 (0.16)</td><td>1.1 (0.16)</td><td>1.1 (0.51)</td><td>1.2 (0.52)</td><td>0.58 (0.53)</td><td>0.34<br>(0.53)</td></tr><tr><td>Progesterone</td><td>P</td><td>0.21<br>(0.072)</td><td>0.66 (0.12)</td><td>0.16<br>(0.034)</td><td>0.74 (0.20)</td><td>0.064<br>(0.025)</td><td>0.48<br>(0.062)</td></tr><tr><td>20α-dihydroprogesterone</td><td>20α-DHP</td><td>1.1 (0.29)</td><td>1.8 (0.28)</td><td>2.0 (0.50)</td><td>2.5 (0.30)</td><td>0.49 (0.12)</td><td>1.1 (0.25)</td></tr><tr><td rowspan="10">Progesterone and<br>metabolites</td><td>5α-dihydroprogesterone</td><td>5α-DHP</td><td>1.0 (0.27)</td><td>1.3 (0.40)</td><td>0.59 (0.25)</td><td>1.2 (0.11)</td><td>0.15<br>(0.045)</td><td>0.19<br>(0.088)</td></tr><tr><td>3α5α-tetrahydroprogesterone (or<br>allopregnanolone)</td><td>3α5α-THP<br>(ALLO)</td><td>0.26<br>(0.056)</td><td>0.19<br>(0.021)</td><td>0.40 (0.15)</td><td>0.47<br>(0.032)</td><td>0.12<br>(0.023)</td><td>0.22<br>(0.030)</td></tr><tr><td>3β5α-tetrahydroprogesterone</td><td>3β5α-THP</td><td>0.026<br>(0.0051)</td><td>0.040<br>(0.0078)</td><td>0.029<br>(0.014)</td><td>0.027<br>(0.017)</td><td>0.011<br>(0.0060)</td><td>0.035<br>(0.011)</td></tr><tr><td>3α5β-tetrahydroprogesterone</td><td>3α5β-THP</td><td>0.020<br>(0.019)</td><td>0.025<br>(0.010)</td><td>0.10<br>(0.057)</td><td>0.032<br>(0.0097)</td><td>-</td><td>-</td></tr><tr><td>3α5α20α-hexahydroprogesterone</td><td>3α5α20α-HHP</td><td>0.037<br>(0.038)</td><td>0.050<br>(0.024)</td><td>0.33<br>(0.028)</td><td>0.27<br>(0.011)</td><td>-</td><td>0.10<br>(0.058)</td></tr><tr><td>11-deoxycorticosterone</td><td>DOC</td><td>0.16<br>(0.054)</td><td>0.25<br>(0.071)</td><td>0.13<br>(0.054)</td><td>0.28<br>(0.052)</td><td>0.032<br>(0.0090)</td><td>0.16<br>(0.032)</td></tr><tr><td>3α5α-tetrahydrodeoxycorticosterone</td><td>3α5α-THDOC</td><td>0.12<br>(0.046)</td><td>0.74 (0.11)</td><td>0.085<br>(0.012)</td><td>0.74 (0.18)</td><td>0.12<br>(0.080)</td><td>1.2 (0.17)</td></tr><tr><td>Corticosteroids and<br>metabolites</td><td>3β5α-tetrahydrodeoxycorticosterone</td><td>3β5α-THDOC</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td></td><td>Corticosterone</td><td>Corticosterone</td><td>-</td><td>74.4 (19.7)</td><td>-</td><td>49.8 (25.4)</td><td>-</td><td>9.1 (13.4)</td></tr><tr><td></td><td>11-deoxycortisol</td><td>11-deoxycortisol</td><td>-</td><td>8.0 (2.5)</td><td>-</td><td>8.0 (1.7)</td><td>-</td><td>5.7 (2.5)</td></tr><tr><td></td><td>Cortisol</td><td>Cortisol</td><td>-</td><td>168.5<br>(24.3)</td><td>-</td><td>173.4<br>(30.7)</td><td>-</td><td>68.4<br>(38.3)</td></tr><tr><td></td><td>Cortisone</td><td>Cortisone</td><td>-</td><td>9.2 (2.0)</td><td>-</td><td>19.0 (6.4)</td><td>-</td><td>2.9 (2.3)</td></tr><tr><td rowspan="10">Androgens and<br>metabolites</td><td>Dehydroepiandrosterone</td><td>DHEA</td><td>2.3 (1.0)</td><td>8.5 (1.7)</td><td>1.6 (0.43)</td><td>6.8 (2.9)</td><td>1.1 (0.92)</td><td>2.6 (2.4)</td></tr><tr><td>Dehydroepiandrosterone sulfate</td><td>DHEAS</td><td>1.2 (1.0)</td><td>7.5 (2.7)</td><td>3.8 (3.4)</td><td>8.8 (4.8)</td><td>1.2 (1.2)</td><td>4.6 (3.7)</td></tr><tr><td>Androstenediol</td><td>ADIOL</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Androstenedione</td><td>ADIONE</td><td>0.26<br>(0.077)</td><td>0.47<br>(0.079)</td><td>0.23<br>(0.086)</td><td>0.34 (0.10)</td><td>0.050<br>(0.034)</td><td>0.090<br>(0.071)</td></tr><tr><td>Testosterone</td><td>T</td><td>0.24<br>(0.067)</td><td>0.52 (0.11)</td><td>0.38 (0.11)</td><td>0.34 (0.21)</td><td>0.18<br>(0.043)</td><td>0.35<br>(0.090)</td></tr><tr><td>5α-dihydrotestosterone</td><td>5α-DHT</td><td>0.069<br>(0.010)</td><td>0.085<br>(0.014)</td><td>0.056<br>(0.0025)</td><td>0.12<br>(0.011)</td><td>0.062<br>(0.0098)</td><td>0.080<br>(0.027)</td></tr><tr><td>3α5α-tetrahydrotestosterone</td><td>3α5α-THT</td><td>0.074<br>(0.012)</td><td>0.20<br>(0.030)</td><td>0.050<br>(0.017)</td><td>0.20<br>(0.058)</td><td>0.043<br>(0.014)</td><td>0.15<br>(0.057)</td></tr><tr><td>5α-dihydroandrostenedione</td><td>5α-DHADIONE</td><td>-</td><td>0.071<br>(0.026)</td><td>0.0050<br>(5.0E-04)</td><td>0.077<br>(0.058)</td><td>-</td><td>0.026<br>(0.013)</td></tr><tr><td>Etiocholanolone</td><td>Etiocholanolone</td><td>0.0075<br>(0.0035)</td><td>0.35 (0.14)</td><td>0.17<br>(0.046)</td><td>0.21<br>(0.075)</td><td>0.0050<br>(0.013)</td><td>0.39<br>(0.036)</td></tr><tr><td>Epiandrosterone</td><td>Epiandrosterone</td><td>0.042<br>(0.030)</td><td>-</td><td>0.063<br>(0.020)</td><td>-</td><td>0.039<br>(0.020)</td><td>-</td></tr><tr><td rowspan="2">Estrogens</td><td>Estrone</td><td>E1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>17β-Estradiol</td><td>E2</td><td>0.019<br>(0.0065)</td><td>0.0050<br>(0.017)</td><td>0.020<br>(0.0064)</td><td>0.020<br>(0.011)</td><td>0.0060<br>(0.0057)</td><td>0.012<br>(0.016)</td></tr></tbody></table>

(determined using Dixon's test) were excluded from the analysis.

For qPCR of slice culture material, expression levels were first normalized to expression levels of GAPDH by subtracting CT values. Normalized CT values were taken to represent expression levels on a log scale and were used directly for statistical analysis. t-tests were used to compare marker gene expression levels between untreated and vehicle-treated slices. A linear mixed model (LMM) with patient ID as a random effect was used to detect overall differences. p-values from the LMM (Wald chi-square test) were adjusted for multiple testing correction using the Benjamini-Hochberg method. When significant, Dunnett's post-hoc comparisons were carried out where expression for each condition was compared to the vehicle condition. For each patient and each well with steroids treatment, the change in expression was calculated compared to the vehicle condition. The mean and standard error of these values were plotted.

Statistical analysis was carried out in the statistical programming environment R using packages 'car', 'nlme' and 'multcomp'.

## 3. Results

### 3.1. Steroid levels in SN and PFC in PD at different Braak-LB stages

Significant levels of the precursor steroid pregnenolone suggest that neurosteroid synthesis may take place in the SN and PFC. Progesterone levels were in the low nanomolar range and were not significantly different between brain regions and disease states. However, several reduced metabolites of progesterone were found to be significantly different between CTR and both PD stages in SN. 5α-DHP levels were significantly altered (F(2,10) = 15; *p* = 0.0010, ANOVA; *p* = 0.0087 after multiple-testing correction). In particular, 5α-DHP levels were found to be significantly reduced in PD6 compared to PD4 (4.2 fold; *p* = 7 × 10⁻⁴) and 5α-DHP in PD6 was also 4.1-fold reduced compared to the CTR group, but this change was not statistically significant (Fig. 1A). Significant changes of ALLO were also found (F(2,10) = 32; *p* = 4.6 × 10⁻⁵, ANOVA; *p* = 0.0011 after correction) in SN. ALLO level was increased in PD4 compared to CTR (2.8 fold; *p* = 2.0 × 10⁻⁵) and
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Steroid measurements in substantia nigra and prefrontal cortex of PD patients and control donors. Progesterone metabolites levels (ng/g = nanogram/gram of tissue analysed) in substantia nigra (A, B, C) and prefrontal cortex (D, E, F) measured by Gas Chromatography-Mass Spectrometry (GC/MS). CTR: control donors; PD4: PD patients in early neuropathological stages (Braak-LB stages 3–4); PD6: PD patients in late neuropathological stages (Braak-LB stages 5–6); 5α-DHP: 5α-dihydroprogesterone; ALLO: allopregnanolone; 3α5α20α-HHP:3α5α20α-hexahydroprogesterone; Circles indicate female donors; triangles indicate male donors. ***p < 0.001.</figcaption></figure>

reduced in PD6 compared to PD4 (2.2 fold; $p = 1.1 \times 10^{-4}$) (Fig. 1B). The level of 3α5α20α-hexahydroprogesterone (3α5α20α-HHP) was also significantly altered ($F(2,10) = 21$; $p = 2.4 \times 10^{-4}$, ANOVA; $p = 0.0030$ after correction), being increased in PD4 compared to CTR (3.1 fold; $p = 1.3 \times 10^{-4}$), similarly to ALLO (Fig. 1C). Note that one male PD4 patient's measurements for these three steroids were excluded for being high-valued outliers in SN.

PFC showed a similar pattern for 5α-DHP, ALLO and 3α5α20α-HHP levels as compared to SN. 5α-DHP level was reduced in PD6 compared to both PD4 and CTR (3.8 and 4.3 fold, respectively), but without reaching statistical significance (Fig. 1D). Likewise, similar changes were found for ALLO in PFC as compared to the SN, but again ANOVA analysis showed no significant differences. ALLO levels did show similar fold changes to those in SN, comparing PD4 compared to PD6, (3.9 fold; n.s.) and comparing PD4 and CTR (2.5 fold increase in PD4, n.s.) (Fig. 1E). As in the SN, 3α5α20α-HHP level was altered in PFC ($F(2,10) = 24$; $p = 1.5 \times 10^{-4}$, ANOVA; $p = 0.0032$ after correction) and was increased in PD4 compared to CTR (4.2 fold; $p = 9.4 \times 10^{-4}$) and to PD6 (21-fold, $p = 1.4 \times 10^{-4}$) (Fig. 1F). One male PD4 patient's measurement for 3α5α20α-HHP was excluded as a high-valued outlier.

No statistically significant changes were found for other steroids analysed in either SN and PFC. The data for all the targeted steroids are given in Table 2 and statistical analyses are presented in Supplementary Table 2.

Because the patterns of steroid level changes for 5α-DHP, ALLO and 3α5α20α-HHP appeared to be similar between SN and PFC, we tested the correlations of values for these three steroids (Supp. Fig. 2). One male PD4 donor was excluded from the preceding analysis as a high-valued outlier for all three steroids in SN, and 3α5α20α-HHP in PFC; correlations were tested with and without this donor. In all cases the values in SN and PFC showed significant correlations (Supp. Fig. 2).

## 3.2. Human post-mortem slice cultures

Many neurons were still viable in the PFC slices of both CTR and PD at 5 days in culture medium with ethanol, the vehicle used for steroid administration (Supp. Fig. 1A, B). In order to detect whether vehicle could affect cells viability, gene expression was measured by qPCR in CTR and PD slices of PFC incubated with or without ethanol. The apoptotic markers APAF1 and p75, the cytoskeletal protein vimentin, and the cell-type markers GFAP (glia fibrillary acidic protein) for astrocytes, RBFOX3 (RNA Binding Protein, Fox-1 Homolog 3) for neurons and HLA (human leukocyte antigen) for microglia were measured. No significant differences were found in the RNA expression of these markers in CTR or PD tissue with or without ethanol (Supp. Fig. 1C).

## 3.3. Effect of steroid treatment on CTR slices

In CTR slices, 5α-DHP treatment did not induce any significant mRNA expression changes. ALLO treatment caused significant changes in mRNA expression of insulin growth factor (IGF1) ($\chi^2(4) = 22.9$; $p = 1.3 \times 10^{-4}$; $p = 0.012$ after multiple testing correction) and glutaminase (GLS; glutamate synthetic enzyme) ($\chi^2(4) = 20.5$; $p = 3.9 \times 10^{-4}$; $p = 0.012$ after correction).

At low physiological concentrations (1 nM and 10 nM), ALLO decreased GLS (1.4-fold; $p = 0.0004$ and 1.3-fold; $p = 0.007$, respectively) while 1 nM ALLO decreased expression of IGF1 (2.5 fold; $p = 0.0003$) (Fig. 2A).

DHEAS affected expression of the anti-apoptotic molecule APAF1 ($\chi^2(4) = 18.7$; $p = 8.9 \times 10^{-4}$; $p = 0.021$ after correction) and the neurotrophin receptor P75 ($\chi^2(4) = 21.2$; $p = 2.9 \times 10^{-4}$; $p = 0.012$ after correction). At low physiological (1 nM and 10 nM) and high physiological concentration (100 nM), DHEAS significantly reduced RNA
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effects of steroids on gene expression in slice cultures of human post-mortem PFC from control (CTR) donors and Braak PD stage 6 (PD6) donors. A) Effect of allopregnanolone (ALLO) on GLS and IGF1 gene expression in CTR PFC slices; B) Effect of DHEAS on APAF1 and p75 gene expression in CTR PFC slices. ALLO treatment reduced expression of glutamate synthetic enzyme GLS and neuroprotective molecule IGF1 while DHEAS treatment reduced expression of apoptotic genes. C) Effect of 5α-DHP on gene expression of GAD1, GLAST1 and TGFB in PD6 PFC slices. D) Effect of allopregnanolone (ALLO) on GLAST1 gene expression, 5α-DHP treatment increased gene expression of the glutamate reuptake effector GLAST1 and the neuroprotective molecule for dopaminergic neurons TGFB. E, F) Effects of DHEAS on gene expression in PD6 PFC slices. Gene expression changes after DHEAS treatment of (E) GAD1, GFAP, GLS, and GLUL; (F) HLA, MBP, RFOXB3 and TGFB. DHEAS treatment at high concentrations reduced gene expression of glial cell and neuron markers and also enzymes involved in the synthesis of neurotransmitters, and TGFB. Circles indicate female donors; triangles indicate male donors. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001.</figcaption></figure>
expression of P75 (1.6, 1.9 and 1.7 fold and p = 0.01, p = 0.003, p = 0.0008, respectively) and reduced APAF1 expression at 1 nM (1.6 fold; p = 0.02) and at 100 nM (1.7 fold; p = 0.007) (Fig. 2B).

### 3.4. Effects of steroids treatment on PD slices

In PD4 slices, 5α-DHP, ALLO and DHEAS caused no significant changes in RNA expression compared to the vehicle control for all the analysed genes.

In PD6 slices, however, all three steroids had effects on gene expression. 5α-DHP affected expression of GAD1 (glutamate decarboxylase 1) ($\chi^2(4) = 21.3$; $p = 2.7 \times 10^{-4}$; $p = 0.003$ after multiple testing correction), GLAST1 (glutamate/aspartate transporter) ($\chi^2(4) = 27.6$; $p = 1.5 \times 10^{-5}$; $p = 0.0005$ after correction) and TGFB (transforming growth factor beta) ($\chi^2(4) = 14.2$; $p = 0.0068$; $p = 0.043$ after correction) (Fig. 2C). GAD1 expression was significantly decreased at 100 nM (1.4 fold; $p = 0.027$), while GLAST1 was increased at all concentrations. TGFB expression was significantly increased at 0.1 nM and 10 nM (1.5 fold, $p = 0.001$; 1.4 fold, $p = 0.026$ respectively) (Fig. 2C).

Moreover, ALLO had an overall effect on GLAST1 expression ($\chi^2(4) = 16.2$; $p = 0.0028$; $p = 0.019$ after correction) with a tendency to increased expression at 10 and 100 nM, although these changes were not significant in post-hoc tests (Fig. 2D).

In PD6 slices, DHEAS decreased expression of GAD ($\chi^2(4) = 22.6$; $p = 1.5 \times 10^{-4}$; $p = 0.003$ after multiple testing correction), GFAP ($\chi^2(4) = 20.3$; $p = 4.4 \times 10^{-4}$; $p = 0.004$ after correction), GLS ($\chi^2(4) = 19.9$; $p = 5.2 \times 10^{-4}$; $p = 0.004$ after correction), GLUL ($\chi^2(4) = 60.5$; $p = 2.3 \times 10^{-12}$; $p = 1.6 \times 10^{-10}$), HLA ($\chi^2(4) = 26.0$; $p = 3.2 \times 10^{-5}$; $p = 0.0007$ after correction), MBP ($\chi^2(4) = 21.1$; $p = 3.0 \times 10^{-4}$; $p = 0.003$ after correction), RBFOX3 ($\chi^2(4) = 14.8$; $p = 0.0050$; $p = 0.036$ after

correction) and TGFB ($\chi^2(4) = 13.9$; $p = 0.0077$; $p = 0.044$ after correction), especially at 10 nM (low physiological concentration) and at 100 nM (a high physiological concentration) (Figs. 2E, F). The strongest effect was on GAD1 (2.4 fold reduction at 100 nM) while the maximum reductions for the other genes were 1.3–1.5 fold.

All statistical test results for steroid effects in slice cultures are given in Supplemental Table 3.

### 4. Discussion

In this study, we have carried out for the first time extensive steroid profiling in human post-mortem SN and PFC, areas involved in the early and late stages of the neuropathological process in PD, respectively. Using Braak Lewy-body staging (Braak et al., 2003), we were able to distinguish PD patients in the initial and in late stages of the disease. Among the 27 measured steroids, we found that three progesterone metabolites are specifically affected in different ways in these neuro-pathological stages. All the other steroidogenic pathways, including the synthesis of pregnenolone, glucocorticoids, androgens, estrogens and all their precursors and metabolites, were not affected at either stage of the disease. In particular, upregulation of ALLO and its 20α-reduced metabolite 3α5α20α-HHP was found at the early disease stage (PD4), with downregulation of all three progesterone metabolites, 5α-DHP, ALLO and 3α5α20α-HHP occurring at the late stage (PD6). These three steroids are highlighted in the schematic representation of steroidogenesis in humans shown in Fig. 3, among all the other steroids measured in this study in post-mortem brain tissues. These disease-stage dependent changes happen most clearly in the SN, an area affected early in the disease, and to a lesser extent in the PFC, which is affected later. This suggests an upregulation of ALLO synthesis when symptoms of the

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Overview of the steroids measured by GC/MS and their synthesis and metabolism pathways. The abbreviated names of the steroidogenic enzymes are in red. The three reduced metabolites of progesterone which were found to have altered levels in substantia nigra (SN) and/or pre-frontal cortex (PFC), namely 5α-DHP, ALLO and 3α5α20α-HHP, are highlighted with boxes. Arrows to light grey boxes indicate metabolites that were upregulated at PD4 (early stage PD) compared to controls and arrows to dark grey boxes indicate those that were downregulated at PD6 (late stage PD) compared to PD4. Steroids in italics were not measured. (3α5β-THP and etiocholanolone were measured but are not shown for simplicity). 17α-OH-PREG: 17α-hydroxypregnenolone, 17α-OH-PROG: 17α-hydroxyprogesterone. For other steroid abbreviations, see Table 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
disease start followed by downregulation of 5α-DHP and ALLO synthesis with disease progression. We have previously found that 5α-DHP and ALLO levels were reduced in the CSF of PD patients in clinically advanced stages (di Michele et al., 2003), and a significant decrease in 5α-DHP levels was also observed in the striatum of rats injected with 6-hydroxydopamine (Melcangi et al., 2012), suggesting that progesterone metabolism is similarly affected in this experimental model of Parkinson's disease.

The pattern of changes that we saw in these three steroids in SN was also reflected in PFC, although it did not reach significance there for 5αDHP and ALLO, probably due to the small sample sizes. This similarity suggests it is likely that these changes are occurring globally throughout the brain. The similarities in steroid regulation between the two areas are supported by the significant correlations of steroid concentrations between the two areas.

One male patient at PD4 was excluded as a high-valued outlier for several readings of these three steroids. It is not clear why this patient had such unusually high levels of progesterone derivatives. Inclusion of this patient's outlying values would result in a loss of significance, due to the increase in variability, although the high values agree with the overall pattern of increased levels of progesterone derivatives at PD4.

Limitations of this study are the small group sizes and especially that the PD6 groups were unbalanced with respect to sex in both the GCMS and slice culture experiments, both being predominantly male. In particular the decreases in progesterone metabolites at late stage Parkinson's are observed in this group and we cannot exclude that the reductions in progesterone metabolites seen at PD6 are partly due to the preponderance of males in this group. However, no significant differences were detected between control donor males and females in levels of progesterone, 5α-DHP, ALLO or 3α5α20α-HHP, although this may be due to limited statistical power. Possibly, control levels of 5α-DHP may be slightly higher in females in SN (Fig. 1), but no baseline differences are apparent in the case of ALLO or 3α5α20α-HHP, and so the differences we detected in the levels of these steroids are unlikely to be due to gender bias. Furthermore, given the ages of patients and control donors, all the female donors were post-menopausal meaning that serum levels of progesterone and its metabolites were low and similar to the male patients. In males and post-menopausal females serum progesterone levels were found to be very similar and stable with age (Oettel and Mukhopadhyay, 2004).

The variability in the control group and relatively small sample sizes may explain why we did not find a significant reduction of 5α-DHP or ALLO levels in Braak Lewy body stage 6 post-mortem material relatively to the control group as was found in previous studies (di Michele et al., 2003), although we did see a tendency to reduced levels of 5α-DHP in both brain areas analysed. This is consistent with our previous finding of reduced RNA expression of the 5α-DHP synthetic enzyme 5α-R1 in SN from PD patients in late neuropathological stages (Braak 5–6) (Luchetti et al., 2010). These data indicate impaired synthesis of neuroactive steroids during the later stages of neurodegeneration.

Increased levels of ALLO in the early stages may represent a compensatory mechanism to increase neuroprotection and to prevent further loss of dopaminergic transmission. Given its important neuroprotective activity (reviewed in (Di Michele et al., 2013; Wang, 2014)), a decrease of local ALLO concentration at late stages of PD may be a contributing factor to neuronal degeneration in SN. ALLO and progesterone treatment have proven to be neuroprotective for dopaminergic neurons and functionally beneficial in animal models of PD (Adeosun et al., 2012; Litim et al., 2017; Nezhadi et al., 2016) and also AD (Sun et al., 2012).

The ALLO metabolite 3α5α20α-HHP, which showed similar changes in PD to ALLO, can modulate GABA<sub>A</sub> receptors in a similar way to ALLO although with less efficacy (Belelli et al., 1996). This metabolite is formed by 20α-reduction of ALLO and the changes in its abundance likely reflect those of ALLO itself. However, since 20α-reduction is reversible, interconversion of 3α5α20α-HHP with ALLO may be an

additional mechanism to buffer ALLO levels. Further investigation of the potential neuroprotective effects of this compound is warranted.

In order to determine possible effects of steroids in the areas affected in PD in different stages of the disease, we used a unique post-mortem human brain slice culture assay. We have previously established that many cells in the cultured slices remain viable for long periods in vitro (Qi et al., 2018; Verwer et al., 2002b; Verwer et al., 2003; Wu et al., 2008). PFC was chosen because this is the last brain area that generates in PD (Braak et al., 2003), potentially allowing any beneficial effects of steroids to be detected. Here, we show that treatment of post-mortem human PD6 brain slices in short-term cultures with 5α-DHP or ALLO (which were found downregulated in PD6 by GC/MS) increases gene expression of GLAST, a molecule which removes glutamate from extracellular space protecting the neurons from excitotoxicity (Sheldon and Robinson, 2007). Moreover, 5α-DHP increases gene expression of TGFB which has shown to have a neuroprotective effect on dopaminergic cells in mouse models of PD (Roussa et al., 2009; Tesseur et al., 2017). Therefore, our data indicate that reduced metabolites of progesterone are potentially neuroprotective in the last stages of the disease. ALLO has potential as a clinically applicable neuroprotective agent (Hernandez et al., 2020) and another ongoing trial (https://clinicaltrials.gov/ct2/show/NCT03748303) may give insight soon into its possible effectiveness.

Slice cultures were also incubated with DHEAS, since we previously found a change in its steroid synthetic enzyme SULT2B1 in SN (Luchetti et al., 2010). However, here we found that DHEAS concentrations were similar between CTR and PD patients in SN and PFC. DHEAS is neuroprotective and an NMDA modulator and could help to regulate the imbalance of GABA and glutamate signalling present in PD (Di Michele et al., 2013). Interestingly, DHEAS decreased gene expression of the apoptotic molecules such as APAF1 and p75 in CTR slices further supporting the neuroprotective effect of DHEAS widely described in the literature (reviewed in (Maninger et al., 2009)). Surprisingly, in PD6 slices, gene expression of GABA synthetic enzyme GAD and glutamate synthetic and metabolic enzymes (GLS and GLUL respectively), myelin basic protein and important astrocyte, microglia and neuronal cell markers (GFAP, HLA and RBFOX3 respectively) was inhibited by DHEAS, especially at high concentrations. This suggests that DHEAS may, unexpectedly, have a negative effect on cell health specifically in late-stage PD. Notwithstanding the general neuroprotective action of DHEAS, it has inhibitory effects on the GABA<sub>A</sub> receptor and potentiates the NMDA receptor (Maninger et al., 2009), thus potentially exacerbating excitotoxicity.

In light of this, the reduced RNA levels of SULT2B1 found before in the PD SN (Luchetti et al., 2010) might be seen as a compensatory mechanism to protect SN from degeneration in later stages of PD. The protective mechanism would be to reduce the conversion of DHEA in the brain to a harmful excitatory neurosteroid and possibly to allow DHEA to serve as a substrate for the production of protective androgens and estrogens. Further studies are needed to test this hypothesis.

In summary, with the use of precise steroid profiling by mass spectrometry, we have found a specific upregulation of ALLO synthesis at the early stage of PD, especially in the SN, followed by a downregulation of ALLO and 5α-DHP synthesis at later stages. Incubation with 5α-DHP and ALLO induced an increase of some neuroprotective molecules in human PFC organotypic culture in PD. These effects were evident only in the late neuropathological stages. Overall, these data suggest i) an endogenous protective compensatory effect of ALLO at early stage of PD, and ii) that therapeutic application of either 5α-DHP and ALLO, or drugs having the same mechanism of action, or molecules stimulating the synthesis of these neuroactive steroids in the brain, could induce neuroprotective effects, especially when their production is impaired as in advanced neurodegenerative stages of PD.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2023.106169.
CRediT authorship contribution statement

**Sabina Luchetti:** Conceptualization, Methodology, Investigation, Funding acquisition, Formal analysis, Writing – original draft. **Philippe Liere:** Methodology, Investigation, Writing – review & editing. **Antoine Pianos:** Investigation. **Ronald W.H. Verwer:** Methodology. **Arja Sluiter:** Investigation. **Inge Huitinga:** Resources. **Michael Schumacher:** Supervision, Writing – review & editing. **Dick F. Swaab:** Supervision, Writing – review & editing, Resources. **Matthew R.J. Mason:** Formal analysis, Writing – review & editing.

Declaration of Competing Interest

None.

Data availability

Data will be made available on request.

Acknowledgments

In memory of the first author, Sabina Luchetti, who sadly passed away before the manuscript could be submitted.

The authors would like to thank, R. Balesar, B. Fisser, for technical assistance. We thank the Netherlands Brain Bank for providing the human material. This work was supported by The Internationala! Parkinson Fonds (now Stichting ParkinsonFonds (IPF 05012012).

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