Glucocorticoids modulate expression of perineuronal net component genes and parvalbumin during development of mouse cortical neurons

Glucocorticoids modulate expression of perineuronal net component genes and parvalbumin during development of mouse cortical neurons | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Glucocorticoids modulate expression of perineuronal net component genes and parvalbumin during development of mouse cortical neurons Liang Yue , View ORCID Profile Michael T Craig , View ORCID Profile Brian J Morris doi: https://doi.org/10.1101/2025.02.12.637910 Liang Yue 1 School of Psychology and Neuroscience, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Joseph Black Building , Glasgow, G12 8QQ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael T Craig 1 School of Psychology and Neuroscience, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Joseph Black Building , Glasgow, G12 8QQ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael T Craig Brian J Morris 1 School of Psychology and Neuroscience, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Joseph Black Building , Glasgow, G12 8QQ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Brian J Morris For correspondence: brian.morris{at}glasgow.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Severe prenatal maternal stress is a risk factor for schizophrenia in offspring. Since parvalbumin-containing GABAergic interneuron function in cortex and hippocampus is compromised in schizophrenia, and perineuronal nets (PNNs) facilitate the functioning of these cells, we tested the hypothesis that glucocorticoids, as stress mediators that can access the foetal compartment, might influence the expression of PNN component genes. In cultured mouse cortical neurons, we detected effects of hydrocortisone on many PNN component genes, via diverse mechanisms. A rapid (<4h), glucocorticoid receptor (GR)-mediated suppression of neurocan and hyaluronan synthase ( Has ) 1 and 3 mRNAs was observed at 7 days in vitro (DIV), whereas at 14DIV, brevican and versican expression was reduced by hydrocortisone without GR involvement, while GR inhibition elevated Has1 and Has2 mRNA levels and suppressed aggrecan mRNA levels. Tenascin R expression was rapidly suppressed by hydrocortisone at 7DIV but not at 14. At 21DIV, PNN component gene expression had become insensitive to hydrocortisone, although parvalbumin expression was reduced after 24h but not 4h exposure. Additionally, effects on protein levels were observed that were sometimes consistent with the mRNA changes (e.g. Has3, Gad1) and sometimes unrelated to them (e.g. elevated TnR levels at 14DIV after glucocorticoid receptor antagonism). We found that hydrocortisone could directly inhibit proteasome activity, potentially explaining the ability of hydrocortisone to increase Has2 levels. As expected from these results, the overall structure of the PNN was compromised by hydrocortisone exposure, with the length of proximal dendrite covered by PNN being reduced. Overall, the data demonstrate a complex and profound, but developmental stage-dependent, regulation of PNN component gene expression by glucocorticoids. This may contribute to the action of severe prenatal or perinatal stress to increase schizophrenia risk. Introduction Perineuronal nets (PNNs) are complex structures composed of extracellular matrix molecules, covering the somata, dendrites and proximal axon segments of distinct neuronal populations ( Fawcett et al., 2019 ), mainly enveloping fast-spiking GABAergic parvalbumin ( Pvalb )-expressing interneurons in cortical and hippocampal areas ( Celio & Chiquet-Ehrismann, 1993 ). Their formation in the developing brain is associated with the onset of Pvalb expression, the maturation of the Pvalb -expressing cells, and the closure of the critical period of cortical plasticity ( Morishita et al., 2015 ; Pizzorusso et al., 2002 ; Willis et al., 2022 ). Once formed, the PNNs are believed to enable fast-spiking activity of these cells by restricting ion movements immediately extracellular to the cell membrane ( Fawcett et al., 2019 ; Morawski et al., 2015 ), while also protecting the cells from oxidative stress ( Morishita et al., 2015 ) and consolidating afferent synapse architecture ( Ferrer-Ferrer & Dityatev, 2018 ). The key components of PNNs are the members of the family of chondroitin sulphate proteoglycans (CSPGs), including aggrecan ( Acan ), brevican ( Bcan ), neurocan ( Ncan ), versican ( Vcan ) and phosphacan ( Ptprz1 ), which are essential for PNN function and structure (Deepa et al., 2006). Hyaluronan (HA), which forms the backbone of PNNs, binding to the CSPGs, is synthesised by cell membrane hyaluronan synthase (Has) enzymes, and extruded into the extracellular space. The structure is stabilised by hyaluronan and proteoglycan link proteins (Haplns), and by cross-linking by Tenascin R ( Fawcett et al., 2019 ). The cellular origins of these different components is not always clear, but it is likely that some are synthesised in neurons and others in astrocytes ( Irala et al., 2024 ; John et al., 2006 ), suggesting that a complex regulatory framework must be required for effective coordination of the synthesis of the different constituent proteins in different cells. During development, PNNs are susceptible to external stressors. Reduced PNN staining intensity and density were found in various brain areas, including prefrontal cortex (PFC), basolateral amygdala ( Allgäuer et al., 2023 ; Santiago et al., 2018 ) and hippocampus ( Riga et al., 2017 ) in rodents after exposure to early life stress. Consistent evidence has also demonstrated downregulated levels of CSPG molecules after exposure to stress ( Koskinen et al., 2020 ; Li et al., 2024 ). PNNs are disrupted as part of the dysfunction of Pvalb -expressing neurons in schizophrenia. Alongside the reduced expression of PVALB and the GABA synthetic enzyme GAD1 /GAD67 in PFC, hippocampus and thalamic reticular nucleus in schizophrenia ( Gonzalez-Burgos et al., 2015 ; Hoftman et al., 2015 ), PNN structure is also compromised ( Enwright et al., 2016 ; Mauney et al., 2013 ; Pantazopoulos et al., 2021 ; Steullet et al., 2018 ). Maternal experience of severe stress (such as famine or natural disaster) during pregnancy increases risk of schizophrenia for the offspring in utero ( Mawson & Morris, 2023 ). While the causal mechanisms are unclear, it is known that glucocorticoids (GCs), which are markedly elevated during the maternal stress response, can cross the placenta and access the foetal compartment ( Fowden et al., 2022 ; Gitau et al., 1998 ; Schwartz, 1997 ; Wieczorek et al., 2019 ). Considering the reports that early life stress could influence PNN development, we considered the hypothesis that GCs might affect the expression of PNN component genes, and hence disrupt the formation or maintenance of PNNs and increase schizophrenia risk. In humans, the main GC is cortisol, while in rodents, the main GC is corticosterone. The canonical GC mechanism involves binding to an intracellular receptor, either the GC receptor (GR) or mineralocorticoid receptor (MR), nuclear translocation of the steroid-receptor complex, and binding to specific sites, usually in gene promoter regions, resulting in repression or activation of gene expression ( Joëls, 2018 ). These transcriptional effects are slow – a typical time-course would have an onset of altered mRNA levels 8-12h after GC exposure ( De Kloet, 2004 ; Joëls, 2018 ), although occasionally more rapid effects can be observed. The altered levels would then be maintained for more than 24h after the original exposure. The non-genomic effects of GCs, which are thought to involve membrane receptor-mediated actions, not necessarily involving alterations in gene transcription, occur much more rapidly (< 1h) ( Hollos et al., 2020 ; Hynes & Harvey, 2019 ; Joëls, 2018 ; Joëls et al., 2013 ). There is also evidence that some rapid, non-genomic effects of GCs do not involve the canonical GR; but other receptors such as GPR97 ( ADGRG3 ) located on the cell membrane (Y. Q. Ping et al., 2021 ). Thus, we sought to test the hypothesis that elevated GC levels would disrupt PNN formation in developing neuronal networks, and to uncover the underlying mechanisms.. Methods Primary cortical neuronal culture Mouse neurons were cultured as previously described ( Cruise et al., 2000 ; Fuller et al., 2001 ). The brains were removed from C57BL/6 mouse embryos at E17 and washed in ice-cold Hanks Balanced Salt Solution (HBSS). The meninges were removed, the cortical tissues were isolated and transferred into clean ice-cold HBSS. After two more washes in clean ice-cold HBSS, the cortical tissues were transferred into 0.05% trypsin/EDTA (Gibco, 25300054) at 37 °C for 10 min. Following this, DMEM (10% HI horse serum, 1% Penicillin Streptomycin, 1% Glutamax) was added to inactivate the trypsin and tissues were centrifuged at 1500 rpm for 5 min. After removing the supernatant, DMEM (8ml/embryo) was added, and the mixture of DMEM and cortical tissues were transferred into clean neurobasal medium (Gibco, 21103049) with B27 supplement (Gibco, 17504044). The cells were then seeded into 12-well plates precoated with 4 μg/ml poly-D-lysine and 6 μg/ml laminin. Subsequently, neurobasal medium with B27 supplement and clean DMEM medium were added to each well. The ratio of cell suspension medium, clean neurobasal medium and DMEM medium in each well was 0.25/0.25/0.5. After 24 h, 50% medium in each well was replaced with new neurobasal medium with B27 supplement; and then 50% of medium in each well changes with Neurobasal/B27 were made every 4 days for the duration of the cultures. Drug treatment To investigate the effect of glucocorticoids on PNN expression at 7, 14 and 21 days in vitro (DIV), cells were treated with low dose (final concentration 20 nM) or high dose (final concentration 100 nM) hydrocortisone acetate (CORT) for 4h or 24h. These concentrations were chosen to reproduce those achieved in the foetus during moderate or severe maternal emotional stress (see Discussion). In some experiments, the selective GR antagonist, mifepristone/RU486 (20 nM final concentration) was added 30 min prior to CORT treatment. In addition, to examine the pharmacology of GC effects, the selective MR agonist, aldosterone (100 nM final concentration), or the selective GR agonist, fluticasone (50nM final concentration), were tested. Alternatively, cells were also treated with collagen 3 (Cell Guidance Systems, 75nM final concentration), an agonist ligand for the adhesion GPCRs GPR56/97 ( Olaniru et al., 2018 ; Zhu, Luo, et al., 2019 ), to investigate whether the effect of glucocorticoids was mediated through this GPCR family. To test whether mRNA stability was affected, CORT treatment occurred together with 5µg/ml Actinomycin D treatment, to inhibit the mRNA synthesis, 1.5 h, 2 h, 3 h, or 4 h prior to mRNA extraction. mRNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR) The procedure was according to our standard methodology ( Paterson et al., 2006 ; Willis et al., 2021 ). Total mRNA was isolated from the cultured cells using RNeasy mini kits (Qiagen 74106). The RNA quality and concentration were confirmed using spectrophotometry before cDNA synthesis. First strand cDNA was synthesized from mRNA using high-capacity RNA-to-cDNA kit with 10 µl RT Buffer (Applied biosystem, 4387406) and 1 µl enzyme (Applied biosystem, 4387406), in a final volume of 20 μL with appropriate volumes of Nuclease water and mRNA samples based on the results of RNA spectrophotometry. The product was aliquoted and stored at −20 °C for future use. The cDNA quality and concentration were confirmed using spectrophotometry before qPCR method. mRNA levels were measured using SYBRgreen methodology. Samples were run in triplicate on 96-well plates with 1 μl of cDNA samples, 19 µl master mix (Agilent), with cycling conditions of 1 cycle 50 °C for 2 min, 1 cycle 95 °C for 2 min, 40 cycles for 30 s at 95 ° C and 10 s at 60 ° C, followed by a melt curve. Gapdh was employed as house-keeping gene to normalise the gene expression of different targeted primers, except in a single case where the treatment (collagen 3) was found to alter Gapdh expression, where Tbp was used instead. The primers targeted Acan, Bcan, Ncan, Vcan, Ptprz1, Has1, Has2, Has3, TnR, Hapln4, Gad1, Gad2 and Pvalb . Data were analysed using the ΔΔCt method. Primer sequences are provided in Supplementary table 1. Protein extraction and western blot Immunoblotting was performed via our standard procedures ( McNair et al., 2010 ; O’Kane et al., 2003 ). The medium was removed from the wells and 1 ml ice-cold PBS (PH7.4) was added to each well for about 1 minute. Following this, 60 μl RIPA buffer (made up with 50 mM Tris-HCL, 150 mM NaCl, 1% Triton 100, 0.15 SDS, 0.5% sodium deoxycholate and 50 mL dH2O) with 1% protease inhibitor cocktail (Sigma P-8340) and 1% Sodium orthovanadate was added to the wells for 3 min. The wells were then scraped with pipette tips, the contents were transferred to 1.5 mL Eppendorf tubes and centrifuged for 10 min at 4°C, 13,000 rpm. Supernatants were collected and the protein concentration was measured using Bradford Protein Assay (Bradford, 1976). The protein samples (−1.5μ g/μl) were prepared with 4x sample buffer (NuPAGE, Novex, NP0007) and reducing reagent (NuPAGE, Novex, NP0004). Protein samples were denatured at 80 °C for 10 min, and 25 μl/lane added to SDS-PAGE in 4%-12% Bis-Tris gels (NuPAGE, Novex, NP0302BOX), followed by electrophoresis at 200 volts for 1.5 h in chilled running buffer. Protein was then transferred to Invitrogen PVALBDF membranes (Invitrogen, LC2002) in transfer buffer at room temperature running for 1 h at 30 V. Membranes were washed twice in ddH2O and blocked in 0.5% Tween-Tris-buffered-saline (TTBS) with 3% dried milk powder (Marvel) for 30 min at room temperature. After blocking, membranes were incubated with primary antibodies at 4°C overnight in 1% TTBS milk. The following morning, membranes were washed 3 times for 10 min in Tris-buffered-saline (TBS) containing 0.05% Tween 20 (Sigma-Aldrich, T7949) and incubated in horseradish peroxidase (HRP)-conjugated anti-mouse/anti-rabbit secondary antibodies (anti-mouse concentration: 1:10,000; anti-rabbit concentration: 1:6000 with 1% dried milk in TTBS for 1.5 −2 h at room temperature. The antibodies that were used in western blots are listed in Supplementary table 2. Membranes were then washed once with TTBS and washed twice with TBS after incubation of secondary antibodies. Membranes with targeted antibody could be detected by adding chemiluminescent HRP Substrate (Immobilon, Millipore, WBKLS0100) using equal quantities of luminol and peroxide solution. Finally, the membranes were placed into cassette and were captured using PXI4 (Syngene) with varied exposure times depending on the antibodies used. Lectin fluorescence Wisteria floribunda agglutinin (WFA) lectin is widely used as a marker to visualise PNNs ( Härtig et al., 1992 ). For immunofluorescence, cells were fixed at 14 DIV with 4% paraformaldehyde for 30 min. Cells were then permeabilised and blocked with PBS (0.3 M NaCl)/0.25%Triton X-100/10% normal goat serum (NGS) for 1h at room temperature. Following the blocking step, cells were incubated in a humidified chamber with biotinylated WFA diluted in 0.3 M PBS/0.25%Triton X-100/3% NGS at 4°C overnight. In the following day, cells were incubated in streptavidin-conjugated Rhodamine Red-X diluted in 0.3M PBS/3% NGS for 1h in the dark. After incubation in the dark, cells were washed three times in 0.3M PBS and mounted with Vectashield mounting media (Vector Laboratories, H-1200). The slides were finally covered with a coverslip. Images were scanned using a confocal microscope (ZEISS, LSM900) with a 10x objective for counting and 20x objective for presentation. All representative images were captured as a Z-stack (15µ M in depth) using a Z step of 0.50 µM, 20X objective lens, image size 1024×1024 pixels. The images were taken using Zen blue 3.0 software and downloaded with summed intensity zen-stack projection in Zen black system. Image analyses were performed using ImageJ ( Schneider et al., 2012 ). The PNN-covering dendrite length and intensity were measured manually in ImageJ. Proteasome activity All 3 catalytic 20S proteasome activities were measured by detecting the 7-amino-4-methylcoumarin (AMC) fluorescence liberated from the synthetic proteasomal substrates: Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC) for chymotrypsin-like activity, Boc-Leu-Ala-Ala-AMC (Boc-LAA-AMC) for trypsin-like activity and Ac-Glu-Pro-Leu-Asp-AMC (Ac-GPLD-AMC) for caspase-like activity. To recognise whether the activities were related to the proteasome, MG132 was used as an inhibitor for all 3 activities of 20S proteasome. Unconjugated AMC was diluted into appropriate concentrations to create a standard curve which allowed the fluorescence signal to be converted to units of AMC. The released fluorescence was measured every 60 s for 30 min using excitation wavelength 340 nm and emission wavelength 450 nm. Statistical analysis Statistical analysis was conducted using Minitab, with ANOVA as the standard approach after checking for normality of data distribution, with Tukey or Fisher post-hoc tests depending on the F value of the factor in ANOVA. Sample sizes given represent the number of individual culture wells from which mRNA or protein was extracted separately. Samples were nested within the culture from which they derived for statistical analysis. Results Efficiency of amplification was tested for all primer pairs, and was in every case between 90 and 120% (Supplementary Information Figure S1). In addition, preliminary experiments determined that the expression of Gapdh mRNA was not affected by the treatments, consistent with previous studies in the laboratory ( Willis et al., 2021 ), and was therefore an appropriate choice for “housekeeping gene” in these studies, apart from collagen 3 exposure, where Gapdh mRNA levels were affected by the treatment but Tbp mRNA levels did not change, and so was used instead (data not shown). CORT treatment altered the mRNA expression of PNN components during neuronal development period The present study aims to investigate the effect of glucocorticoids on the gene expression of PNN components in mouse cortical neurones. The components tested in the study included CSPGs ( Acan , Bcan , Ncan and Vcan ), HAS ( Has1 , Has2 and Has3 ), Hapln4 (also known as Brain link protein 2/Bral2) and tenascin R ( TnR ). Attempts were also made to detect Hapln 1, Hapln2 and Hapln3 , but they appeared to be below the threshold for reliable detection (Ct’s ∼ 33 or higher). We then studied the effect of exposure at 7, 14 and 21 DIV to low (20nM) or high dose (100nM) CORT and where effects of CORT were observed, we sought to investigate the involvement of GR activation in the actions: mifepristone (100nM) was cotreated CORT. Mifepristone has nanomolar affinity for glucocorticoid receptors, but micromolar affinity for mineralocorticoid receptors (Testas et al.,1983; Galanuad et al., 1984; Cain et al., 2019). The mRNA expression of Vcan was not significantly changed by CORT exposure at 7 DIV (4h: F (2, 43) =1.02, p=0.370; 24h: F (2, 42) =0.42, p=0.66) ( Fig.1 A, D ), or 21 DIV (4h: F (2, 19) =3.00, p=0.085; 24h: F (2, 18) =0.11, p=0.894) after 4h and 24h CORT treatment ( Fig.1 C,F ). Download figure Open in new tab Figure 1. A-F: mRNA expression of Vcan after 4h and 24h exposure to low dose (20 nM) and high dose (100 nM) CORT at 7, 14 and 21 DIV, at 7 and 21 DIV with CORT alone, and at 14 DIV also with mifepristone (20 nM) treatment. Sample numbers: A,D - n=14-16/group; B-F - n=7-8/group. *p<0.05 vs corresponding vehicle group, post-hoc Tukey’s test. Boxes show median and interquartile range, with whiskers from minimum to maximum. However, clear changes were observed at 14 DIV. There was a significant effect of CORT treatment at both 4h (F (2, 46)=3.32, p=0.046) and 24h (F(2, 46)=3.92, p=0.028) ( Fig.1 B,E ). The post-hoc testing revealed that the expression of Vcan decreased after high dose CORT treatment at both 4h and 24h relative to vehicle treatment (4h: veh vs high dose CORT p=0.048, 24h: veh vs high dose CORT p=0.021). The overall effect of mifepristone, independent of whether CORT was absent or present, was not significant at 4h (F(2, 46)=2.37, p=0.131) or 24h (F(2, 46)=0.01, p=0.943). However, the interaction of CORT and mifepristone was significant at 4h (F(2, 46)=4.63, p=0.016) ( Fig. 1B ), but not 24h (F(2, 46)=0.43, p=0.653) ( Fig.1E ), suggesting that mifepristone treatment exacerbated the corticosterone-driven suppression of Vcan expression at 4h (e.g. veh/mifepristone vs veh/veh - p=0.027 Fisher post-hoc test). Hence, glucocorticoids seem to exert a rapid non-GR-mediated suppression of Vcan mRNA levels, and a basal glucocorticoid receptor-mediated enhancement by GCs in the medium, which is rapidly blocked by mifepristone. In short, a rapid suppressive non-GR action and a rapid enhancing GR action on Vcan expression. No significant effect of CORT on Vcan expression was observed at 21 DIV (Supplementary figure S3). There was a tendency for Acan mRNA expression at 7 DIV to decrease after high dose CORT treatment at 4h (F (2, 43)=3.06, p=0.058; high dose VS vehicle, P=0.046, post-hoc Tukey test), but not 24h (F(2, 47) =1.32, p=0.277) ( Fig.2 A,C ). No significant effects were detected at 14 ( Fig.1 B,D ) or 21 DIV (Supplementary figure S3) (14 div 4h: F(2, 19)=2.11, p=0.152, 24h: F(2, 21)=2.11, p=0.152; 21 div 4h: F (2, 18)=1. 06, p=0.370, 24h: F (2, 19) =3.49, p=0.085), although the same trend for suppression at 4 but not 24h was noted at 14 DIV. Download figure Open in new tab Figure 2: mRNA expression of Acan (A-E), Bcan (F-K), or Ncan (L-N) after exposure to vehicle (Veh), low dose (20nM) or high dose (100nM) CORT at 7 (A,C,E,F,H,L,M.) or 14 (B,D,G,I,J,K,N), with drug exposure for 4h (A,B,E,F,G,J,L) or 24h (C,D,H,I,K,M,N). 7 DIV experiments with CORT only, n=14-16/group; experiments also with mifepristone (20nM), n=7-8/group; 14 DIV experiments with CORT only, n=6-8/group; experiments also with mifepristone (20nM), n=7-8/group; 21 DIV n=6-8/group. # p<0.05, ### p<0.001 overall effect mifepristone vs vehicle groups (ANOVA); * p<0.05, *** p<0.001 vs corresponding vehicle group, ∼ p<0.05 for comparison shown, (post-hoc Tukey test). Boxes show median and interquartile range, with whiskers from minimum to maximum. Download figure Open in new tab Figure 3: Bcan protein expression after exposure to CORT (20 or 100nM) and/or mifepristone (20nM). Representative results from western blotting are shown after 4h or 24h ( B ) exposure at 14 DIV. ( C,D ) corresponding box and whisker plots showing normalised mean intensity of the band signals after CORT and mifepristone exposure at 14 DIV with 4h ( C ) or 24h ( D ) exposure, for immunoreactive bands at 145 and 155 KDa. (n=46 in total, Veh: veh=7, 20nM=8, 100nM=8; Mifepristone: veh=7, 20nM=8, 100nM =8). Boxes show median and interquartile range, with whiskers from minimum to maximum. When the experiment was repeated to test any influence of mifepristone, no significant effect of CORT was shown with 4h exposure (F (2, 44)=0.25, p=0.779) ( Fig.2 E ), although there was a hint of a suppression as before, (p=0.085 veh without mifepristone vs 20nM CORT without mifepristone, post-hoc Tukey test), but mifepristone overall significantly increased expression of Acan (F (2, 44)=4.18, p=0.048). The interaction of mifepristone and CORT treatment was not significant (F (2, 44)=1.78, p=0.183). The results suggested that there was some basal suppression of Acan expression by GCs, which limited the ability to detect further suppression with CORT application, but which was revealed by mifepristone exposure. Bcan mRNA expression decreased significantly after 4h exposure to low dose and high dose CORT treatment at 14DIV (F (2, 19) =3.62, p=0.048; veh vs low dose: p=0.025, veh vs high dose p=0.035, Fisher post-hoc tests), despite considerable variability in the control group ( Fig.2 G ). No significant changes in gene expression were detected at 7DIV (4h: F (2, 43) =2.80, p=0.072; 24h: F (2, 47) =0.85, p=0.436) ( Fig.2 F ), 14DIV (24h: F (2, 21) =0.53, p=0.596)( Fig 2I ) or 21 DIV (4h: F (2, 18) =1.06, p=0.704) (Supplementary Fig. S3). When the experiment was repeated in the absence or presence of mifepristone, Bcan mRNA levels were again found to be decreased by CORT in the absence of mifepristone (veh vs low dose CORT p=0.039, veh vs high dose CORT p=0.020, Fisher post-hoc tests). There was an overall significant effect of CORT treatment on Bcan mRNA expression at 14DIV after 24h (F (2, 46) =6.85, p=0.003), but not 4h (F (2, 46) =1.19, p=0.316), suggesting that the mRNA expression of Bcan decreased significantly after 24h low dose CORT treatment (veh vs low dose CORT p=0.002 Tukey post-hoc tests) ( Fig. 2J,K ). The results did not show a significant overall effect of mifepristone on Bcan expression, at either 4h (F (2, 46)=0.29, p=0.592) or 24h (F(2, 46)=0.97, p=0.329). These results indicated that at 14DIV, the ability of 20nM CORT to reduce Bcan mRNA levels was very clearly unaffected by the presence of mifepristone ( Fig. 2J,K ). There was a significant interaction of CORT and mifepristone treatment at 4h (F (2,46) =3.63, p=0.036), but not 24h (F(2, 46)=0.51, p=0.603). Mifepristone caused a suppression of Bcan mRNA levels (veh with mifepristone vs veh without mifepristone p=0.018, Fisher post-hoc tests). At 21DIV, the expression of Bcan was not affected by CORT at 4h (F(2, 44)=0.59, p=0.560) or 24h (F(2, 44)=2.78, p=0.075). Moreover, the effect of mifepristone was not significant either, at 4h (F(1,44)=0.01, p=0.936) or 24h (F(1, 44)=1.58, p=0.217). Similarly, the interaction of mifepristone and CORT treatment was not significant with 4h (F(1, 44)=1.24, P=3.000) and 24h (F(1, 44)=0.78, p=0.466) exposure (Supplementary figure 3). Hence, just as with the regulation of Vcan expression, GCs seem to exert a rapid non-GR-mediated suppression of Bcan mRNA levels by CORT. These complex effects on Bcan mRNA suggest a rapid non-GR-mediated suppression by CORT, and a basal GR-mediated enhancement by GCs in the medium, which is rapidly blocked by mifepristone at 14DIV. CORT exposure also showed an ability to suppress Ncan expression. At 7 DIV, a significant effect of CORT on Ncan was detected after 4h ( Fig. 2L ) (F(2,45)=7.66, p=0.002) and 24h (F(2, 45)=4.96, p=0.012), (veh vs low dose CORT at 4h p=0.008) and decreased with both low and high dose treatment at 24h (veh vs low dose CORT p=0.017, veh vs high dose CORT p=0.036, Tukey post-hoc tests). The same reduction was also detected in the presence of mifepristone, with lower expression after 24h low dose CORT (veh vs low dose CORT, p=0.023, Tukey post-hoc tests), but no significant changes with 4h CORT exposure in the presence of mifepristone (veh vs low dose CORT, p=0.869, veh vs high dose CORT, p=0.683, Tukey post-hoc tests). The interaction of mifepristone and CORT treatment was significant after 4h (F(2, 45)=9.79, p<0.001), and 24h (F(2, 45)=2.20, p=0.124), in that expression of Ncan with 4h low dose CORT exposure was increased in the presence of mifepristone compared to in its absence (p=0.024, Tukey post-hoc tests), but decreased for the same comparison after 24h (p=0.006). Additionally, there was an overall effect of mifepristone to decrease Ncan mRNA expression over 24h (F(2, 45)=12.47, p=0.001), but not 4h (F(2,45)=1,06, p=0.310). Overall this suggests a suppressive effect of slightly increasing GC levels at 4h through GRs (mifepristone sensitive), and also not via GRs over a longer time scale (mifepristone insensitive), combined with the basal levels of GCs in the medium tending to enhance Ncan expression (probably genomic in mechanism, since slowly relieved over 24h by mifepristone, resulting in a further decrease in levels). No changes in Ncan expression were detected at 21 DIV ( Fig. 2 O , Supplementary figure 3). Although several Bcan mRNA alterations were observed at 14DIV, the protein expression of Bcan (155kDa) ( Fig.2 K L) (4h: F (2, 26)=1.5, p=0.227) remained unchanged after 4h CORT exposure. With mifepristone cotreatment, no significant changes were observed ( Fig.2 L M) (155kDa/145kDa) protein levels with both 4h and 24h CORT exposure (155kDa: 4h F(2, 47)=0.97, p=0.389, 24h: F(2, 47)=0.77, p=0.469; 145kDa:4h F(2, 47)=2.64, p=0.083, 24h: F(2, 47)=1.10 p=0.342). Moreover, there were no overall effects of mifepristone (155kDa: 4h F(1, 47)=1.54, p=0.221, 24h: F(1, 47)=0.00, p=0.963; 145kDa:4h F(1, 47)=0.21, p=0.652, 24h: F(1, 47)=0.23, p=0.631) and no significant interactions of CORT and mifepristone (155kDa: 4h F(2, 47)=0.27, p=0.765, 24h: F(2, 47)=0.28, p=0.759; 145kDa:4h F(2, 47)=0.38, p=0.686, 24h: F(2, 47)=1.73, p=0.190) detected after 4h and 24h exposure. For phosphacan ( Ptprz1 ), mRNA expression was unchanged after 4h or 24h CORT exposure, with low or high doses, at 7 DIV, 14 DIV and 21 DIV (Supplementary figures S3, S4). Has1 gene expression was not significantly affected at 7 DIV (4h: F (2, 21) =2.02, P=0.161; 24h: F (2, 23) =2.71, p=0.09) ( Fig.4 A ) or 14 DIV (4h: F (2, 8) =0.07, p=0.933, 24h: F (2, 12) =1.75, p=0.223) ( Fig.4 C and data not shown), although there was a trend towards a decrease in Has1 expression at 7 DIV after 4h with 100nM CORT (p=0.068, post-hoc Tukey test). When repeated without or with mifepristone, there was again a tendency towards decreased Has1 mRNA expression with 4h CORT exposure to the lower dose ( Fig.4 A, B ), but there was also a great deal of variability with the higher dose. Overall, including mifepristone groups, there was no significant CORT effect (F (2, 44) =0.204 p=0.959) ( Fig.4 B ). There was however an overall effect of mifepristone (F (2, 44) =6.75, p=0.014), indicating mifepristone significantly increased expression of Has1 , consistent with medium GCs acting to suppress mRNA levels. The mifepristone x CORT treatment interaction was not significant (F (2, 44) =1.78, p=0.183). After long-term (24h) exposure to CORT and mifepristone at 7 DIV, the mRNA levels of Has1 still remained unchanged (F (2, 44) =1.34, P=0.273), and with no overall effect of mifepristone (F (1, 44) =1.29, p=0.263) and no interactions of CORT and mifepristone (F (2, 44) =0.26, p=0.775) were found (data not shown). At 14DIV, no significant effect of CORT was observed after 24h CORT treatment (F(2, 46)=0.88, p=0.422), but there was now a really clear overall effect of mifepristone (F(2, 46)=12.48, p=0.001) ( Fig 4C ). The interaction of mifepristone and CORT treatment was not significant (F(2, 46)=0.35, p=0.708). These results suggested that mifepristone significantly blocked a basal suppression of Has1 expression by glucocorticoids in the culture medium, probably rendering it problematic to reveal a clear additional effect of CORT addition. The results suggested a rapidly relievable effect of glucocorticoids in the culture medium to suppress Has1 mRNA levels at 7 and 14 DIV, an effect attenuated by mifepristone exposure, and slightly enhanced by exogenous CORT exposure. Download figure Open in new tab Figure 4: Effects of CORT on Has gene expression A-C : Has1 mRNA expression; D,E : Has2 mRNA expression, and F-H : Has3 mRNA expression after 4h or 24h exposure to low dose (20nM) or (100nM) high dose CORT at 7 or 14 DIV, in the absence or presence of mifepristone (20nM). (7 DIV, n=6-8/group; 14 DIV, n=7-8/group; I,J : Western blot analysis of Has3 protein levels at 7 and 14 DIV after 4h or 24h exposure to 20nM or 100nM CORT alone ( I ), or without or with mifepristone (20nM) treatment ( J ). n: veh=8, 20nM =8, 100nM =8; Mifepristone: veh=8, 20nM=8, 100nM =8. Example blots are shown, with corresponding quantification below. K : Corresponding western blot analysis of Has3 protein levels exposed to the same doses of COR for 24h. n: veh=12, 20nM CORT=12, 100nM CORT=10. *p<0.05, ***p<0.001 vs corresponding vehicle group, post-hoc Tukey’s test; ∼∼ p<0.01 for comparison shown, post-hoc Fisher’s test; # p<0.05, ## p<0.01, ### p<0.001, overall effect of mifepristone vs corresponding (mifepristone) vehicle group, ANOVA. Boxes show median and interquartile range, with whiskers from minimum to maximum. For Has2 mRNA expression, no overall effect of mifepristone was detected at 7 DIV at 4h (F (2, 44) =2.50, P=0.123), with no significant interaction of mifepristone and CORT treatment (F (2, 44) =0.80, p=0.457) (data not shown). Equally, no alterations of Has2 mRNA levels were observed after longer-term exposure to CORT (F (2, 44) =1.98, p=0.151) at 7 DIV, and no overall effect of mifepristone was detected either (F (1, 44) =1.85, p=0.181) ( Fig.4 D ), with no interactions between CORT and mifepristone (F (1, 44) =0.38, p=0.688. At 14 DIV, the changes in Has2 expression were again not significant 24h after CORT treatment (F (2, 43) =0.36, p=0.701), but the expression of Has2 was significantly affected by mifepristone (F (2, 43) =7.20, p=0.011) ( Fig.4 E ), suggesting increased Has2 expression after blocking GRs. Interactions of mifepristone and CORT treatment were not significant (F (2, 43) =1.46, p=0.246). At 21 DIV, the effect of CORT was not significant at 24h (F (2, 46) =0.25, p=0.783) (Supplementary figure S3). Mifepristone did not have an overall effect on Has2 expression at 24h either (F (1, 47) =0.11, p=0.738). And there was no significant interaction of mifepristone and CORT treatment detected with 24h (F (2, 46) =1.10, p=0.303) exposure. Has3 mRNA expression decreased significantly at 7 DIV after 4h 100nM CORT treatment (F (2,9) =5.40, p=0.038, veh vs high dose CORT, p=0.039, Tukey post-hoc test) ( Fig.4 F ). With mifepristone, at 7 DIV, while there was no significant effect of CORT treatment overall at either 4h (F(2,45)=0.82, p=0.452) or 24h (F(2,45)=1.97, p=0.753) ( Fig.4 G,H ), and the overall effect of mifepristone was not significant either, at 4h (F(2,45)=2.04, P=0.163) or 24h (F(2,45)=2.45, P=0.126) ( Fig.4 G,H ), there was a significant interaction between CORT and mifepristone treatment after 4h (F(2, 45)=3.65, p=0.039), but not 24h (F(2, 45)=0.05, p=0.955), suggesting a tendency for mifepristone to attenuate the suppression of Has3 mRNA levels by exposure to the high dose of CORT (high dose CORT with mifepristone vs high dose CORT without mifepristone p=0.009, Fisher post-hoc test). At 21 DIV, no significant effects of CORT on the expression of Has3 mRNA levels were found at 4h (F(2,47)=1.79, p=0.179) or 24h (F(2, 47)=0.24, p=0.787) (Supplementary figure S3). There was no effect of mifepristone at 4h (F(1, 47)=0.30, p=0.43) or 24h (F(1,47)=2.70, p=0.108). Additionally, no significant interaction of mifepristone and CORT treatment was detected with 24h (F(2,47)=0.30, P=0.743) exposure (Supplementary figure S3). The results imply that, at 7 DIV, a suppressive effect of CORT on Has3 mRNA levels is rapidly relieved by antagonism of GRs. We checked for corresponding protein alterations after CORT exposure, Has3 protein levels decreased significantly after 4h exposure with both low dose and high dose CORT (F(2,17)=11.14, p=0.014 veh vs low dose CORT, p=0.001 veh vs high dose CORT, Tukey post-hoc test) ( Fig 4I ). When re tested with mifepristone, the same trend was observed for CORT in the absence of mifepristone at 4h but not 24h exposure, but no overall CORT effect was found at either 4h (F (2, 47) =0.41, p=0.665) or 24h (F (2, 47) =1.21, p=0.309) of CORT and mifepristone exposure ( Fig.4 J,K ). However, mifepristone significantly increased Has3 protein levels after 4h (F (1, 47) =8.29, p=0.006) ( Fig.4 J ), but not quite significantly after 24h exposure (F (1, 47) =3.38, p=0.073) ( Fig.4 K ). No significant interactions of CORT and mifepristone were observed with either 4h or 24h exposure (4h F (2, 47) =0.51, p=0.603, 24h: F (2, 47) =1.12, p=0.336). The evidence therefore points to a rapid suppression of neuronal Has3 expression at both mRNA and protein levels by GCs via GRs. Expression of Hapln4 mRNA was unaffected by CORT after either dose or treatment time, except for a clear suppression of mRNA levels at 4h (but not 24h) after treatment at 14 DIV that was not attenuated by mifepristone (Supplementary figure S5). However, Hapln4 protein levels were unchanged by CORT exposure at 14 DIV (Supplementary figure S5). The mRNA levels for TnR decreased significantly at 7 DIV after 4h low dose CORT treatment (F (2, 45)=3.28, p=0.049, veh vs low dose CORT p=0.038, Tukey post-hoc tests), but while the same trend was observed at 24h, the effect was not significant (F(2,45)=0.49, p=0.619). This decrease was maintained in the presence of mifepristone after 4h CORT exposure, but the mRNA level decreased with the presence of mifepristone with high dose CORT. There was an overall effect of mifepristone on TnR expression which was significant after 24h (F (2, 45)=9.17, P=0.004), but not 4h (F(2, 45)=1.69, P=0.201), ( Fig.5 D ). The level of mRNA expression with 24h mifepristone and CORT exposure was lower than that in the absence mifepristone. Download figure Open in new tab Figure 5: A-F : mRNA expression of TnR after 4h or 24h exposure to low dose (20nM) or high dose (100nM) CORT at 7, 14 and 21 DIV (B-F), and in the absence or presence of mifepristone (20nM) (A,D). (7 DIV: n=7-8/group; 14 DIV: n=9-11/group; 21 DIV: n=12-14/group). # p<0.05, overall effect of mifepristone vs corresponding (mifepristone) vehicle group, ANOVA. Boxes show median and interquartile range, with whiskers from minimum to maximum. In addition, there was a significant interaction of CORT treatment and mifepristone treatment after 24h (F(2, 45)=5.42, P=0.008) rather than 4h (F(2, 45)=2.51, P=0.095), suggesting a reduction of mRNA levels of veh treatment group after long-term mifepristone exposure (veh without mifepristone vs veh with mifepristone p<0.001, low dose CORT with mifepristone vs low dose CORT without mifepristone p=0.045, Fisher post-hoc tests). There were no significant changes in TnR expression after CORT exposure at 14 DIV (4h: F (2, 12) =1.32, P=0.309; 24h: F (2, 11) =0.33, P=0.729) ( Fig.5 B,E ) or 21 DIV (4h: F (2, 16) =0.01, P=0.990; 24h: F (2, 20) =1.65, P=0.219) after either 4h and 24h treatment ( Fig.5 C,F ). In contrast to the decrease in mRNA expression, protein levels of TnR remained unchanged with 4h or 24h CORT exposure at 7 DIV (Supplementary figure S6). CORT alters GABAergic gene expression As PNNs surround mainly Pvalb-expressing GABAergic interneurons (Gabungcal et al., 2013; Morishita et al., 2015 ), GABA related components, including glutamate decarboxylase ( Gad ) genes and Pvalb w ere also measured in the current study. The expression of Pvalb decreased significantly after 24h high dose CORT treatment at 21 DIV (F(2, 20)=4.72, p=0.023, veh vs high dose CORT p=0.043, Tukey post-hoc tests), but not 4h (F(2, 18)=0.26, p=0.775) ( Fig.6 C,F ). However, there were no significant changes at 7 DIV (4h: F(2, 23)=1.25, p=0.308; 24h: F(2, 22)=0.50, p=0.613) ( Fig.6 A,D ) or 14 DIV (4h: F(2, 23)=0.25, p=0.785; 24h: F(2, 22)=1.31, p=0.292) ( Fig.6 B,E ), after either 4h or 24h CORT treatment. Download figure Open in new tab Figure 6: A-F : mRNA expression of Pvalb after 4h and 24h exposure to low dose (20nM) or high dose (100nM) CORT at 7, 14 and 21 DIV. (7 DIV: n=20 in total, veh=8, low dose=6, high dose=7; 14 DIV: n=28 in total, veh=9, low dose=11, high dose=9; 21 DIV: n=39 in total, veh=14, low dose=14, high dose=12). G-H : mRNA expression of Pvalb at 7 DIV (4h) and 21 DIV (24h), after CORT and mifepristone (20nM) treatment. (n=45 in total, Veh: veh=8, low dose samples=8, high dose samples=7; Mifepristone: veh=8, low dose samples=7, high dose samples=7) *p<0.05 vs corresponding vehicle group, ∼ p<0.05 for comparison shown, post-hoc Tukey’s test; # p<0.05 overall effect of mifepristone vs corresponding vehicle group, ANOVA. Boxes show median and interquartile range, with whiskers from minimum to maximum. When CORT treatment was repeated, either in the absence or presence of mifepristone, the results again showed that at 7 DIV, there was no significant effect of CORT on the expression of Pvalb after 4h exposure (F(2, 43)=0.89, p=0.420) ( Fig. 6 G ). However, there was a significant overall effect of mifepristone (F(1, 43)=12.40, p=0.001) with an increase in expression of Pvalb in cultures exposed to mifepristone. For individual group comparisons, the Pvalb mRNA levels significantly increased with 4h exposure by 20nM CORT with mifepristone compared to 20nM CORT without mifepristone (p=0.022, Tukey post-hoc tests), with the same trend at other CORT doses. This implies a basal GR-mediated suppression of Pvalb mRNA at 7 DIV (despite the low basal levels of expression at this developmental stage). At 21 DIV, there was a once more a significant effect of high dose CORT after 24h exposure, maintained in the presence of mifepristone (F(2,47)=8.66, p=0.001; veh with mifepristone vs low dose CORT with mifepristone p=0.035, veh with mifepristone vs high dose CORT with mifepristone p<0.001, post-hoc Tukey test) ( Fig.6 H ). The overall effect of mifepristone was not significant (F(1,47)=1.88, p=0.178), and the interaction of mifepristone and CORT treatment was not detected with 24h exposure (F(2,47)=0.88, p=0.423), suggesting that the clear suppressive effect of CORT on Pvalb expression was not mediated by GRs. GAD1 expression is also robustly found to be decreased in PFC in schizophrenia ( Gonzalez-Burgos et al., 2015 ; Hoftman et al., 2015 ). When tested either in the absence or presence of mifepristone at 7 DIV, the expression of Gad1 was not significantly changed after 4h CORT exposure (F (2,40)=0.93, p=0.406) ( Fig.7A ). Equally, no changes were observed after 24h exposure (Supplementary figure S7). However, the overall effect of mifepristone after 4h exposure approached significance (F (1,40) =4.01, p=0.053), suggesting a possible suppression of Gad1 expression by GCs in the culture medium that is relieved in the presence of mifepristone. There was no significant interaction between CORT and mifepristone exposure (F (1,40) =4.01, p=0.237). At 14 DIV, after 4h exposure, no significant overall change in Gad1 mRNA levels was found for either CORT (F (2,47) =0.22, p=0.803) or mifepristone (F (1,47) =0.19, p=0.661) ( Fig.7A ). However, there was a significant interaction of CORT and mifepristone (F (2,47) =0.823, p=0.038), with reduction of mRNA levels of Gad1 after high dose (100nM) CORT exposure relative to vehicle in the absence of mifepristone (p=0.045, Fisher post-hoc test) ( Fig. 7 A ). At 21 DIV, with 24h exposure, no effect of either CORT (F (2,46) =0.71, p=0.496) or mifepristone (F (1,46) =0.21, p=0.647) was observed, with no significant interaction between the two treatments (F (2,46) =0.23, p=0.798) ( Fig 7A ). Download figure Open in new tab Figure 7: A,B : mRNA expression of Gad1 (A) and Gad2 (B) after 4h (left and centre graphs) and 24h (right graph) exposure to low dose (20nM) or high dose (100nM) CORT at 7, 14 and 21 DIV. n=7-8/group (GAD1), 6-8/group (Gad2 7 DIV), 9-11/group (Gad2 14 DIV) or 12-14/group (Gad2 21 DIV). C : Immunoblotting with anti-Gad65/67 and anti-Gapdh antisera at 7 DIV (24h) after CORT and mifepristone (20nM) treatment. (n=96 in total: veh: veh=16, low dose CORT=16, high dose CORT=16; Mifepristone: veh=16, low dose CORT=16, high dose CORT=16). * p<0.05 vs corresponding vehicle group (post-hoc Fisher’s test); ∼p=0.053, ## p<0.01, ### p<0.001, overall effect of mifepristone vs corresponding vehicle groups, ANOVA. Boxes show median and interquartile range, with whiskers from minimum to maximum In contrast to the effects of CORT on Gad1 expression, no changes were detected in Gad2 (GAD65) mRNA expression following CORT exposure at 7, 14 or 21 DIV at 4 or 24h (F(2,23)=0.38, p=0.687 (4h); F(2,23)=0.33, p=0.723 (4h); F(2,20)=0.73, P=0.495 (24h) respectively) ( Fig 7B , Supplementary figure S7). Protein levels were also measured based on the mRNA alterations, at 7 and 14 DIV after 24h exposure. At 7 DIV, there were no significant changes of Gad67/65 with 24h CORT exposure, although there was a slight trend towards decreased expression ( Gad67 : 24h F(2, 47)=3.15, p=0.053; Gad65 : 24h: F(2, 47)=2.61, p=0.085) ( Fig.7 C ). However there was a significant overall effect of mifepristone on Gad65/67 protein levels ( Gad67 : 24h F(1, 47)=12.72, p=0.001; Gad65 : 24h: F(1, 47)=13.68, p=0.001), with increased expression at 24h. There were no significant interactions of CORT and mifepristone were detected inn Gad67/65 ( Gad67 : F(2, 47)=0.72, p=0.493; Gad65 : 24h: F(2, 47)=2.61, p=0.085) ( Fig.7 C ). At 14 DIV, neither CORT nor mifepristone had any significant effect on Gad1/Gad67 or Gad2/Gad65 protein levels after 24h exposure (Supplementary Figure S8). These results suggested that CORT had little overt effect on Gad1/Gad67 protein levels, however at 7 DIV, but not later in development, mifepristone exposure revealed a basal suppression of both Gad67 and Gad65 protein, and Gad1 mRNA, by basal levels of GCs in the culture medium. Effect of Collagen 3 (GPR56/97 agonist) and aldosterone (MR agonist) The previous results showed that glucocorticoids could regulate the expression of PNN components, and the changes of Has3, Gad1 , and Pv at 7 DIV could be reversed by the selective GR antagonist, mifepristone. However, the expression of other PNN components which was significantly regulated by glucocorticoids was not reversed by mifepristone. In this case, the effect might be activated via other GC targets rather than via GRs. Therefore, GPR56/GPR97 (ADGRG1/ADGRG3) were considered as a potential alternative pathway for the GC effect, as GCs are agonists at these receptors (GPR97/ADGRG3) (Y.-Q. Ping et al., 2021 ), collagen 3, an agonist of GPR97 and GPR56 ( Luo et al., 2011 ; Olaniru et al., 2018 ; Zhu, Luo, et al., 2019 ) (considering the similarity between GPR97 and GPR56 at the binding site, they are likely to share the same ligands)( Vizurraga et al., 2020 ) was tested on PNN component mRNAs which were affected by GCs but were not sensitive to mifepristone reversal (7 DIV: Ncan , TnR; 14 DIV: Bcan , Vcan , Hapln4 , Gad1 ). The mRNA expression of TnR was significantly upregulated by collagen 3 treatment for 4h at 7DIV (P=0.020, F (1, 23) =6.33) ( Fig 8 A ). Ncan mRNA expression was downregulated by collagen 3 treatment for 4h (P=0.006, F (1, 23) =9.29) ( Fig. 8 A ) at 7DIV. The results indicated that collagen 3 could increase TnR mRNA expression and suppress Ncan mRNA expression rapidly. The former effect is opposite to the effect of GCs, but the effect on Ncan expression is similar, raising the possibility that both collagen 3 and GCs could be acting via the same (non-genomic) GPCR-mediated mechanism in this case. Download figure Open in new tab Figure 8: A . mRNA expression of TnR and Ncan at 7DIV after 4h collagen 3 (75nM) treatment; (n=23 in total, veh=12, collagen 3 treated samples=11). B . mRNA expression of Bcan, Vcan, Hapln4 and Gad1 at 14DIV after 4h collagen 3 (75nM) treatment. C. mRNA expression of Bcan, Vcan, Hapln4 and Gad1 at 14DIV after 4h treatment with aldosterone (100nM) or fluticasone (50nM). (n=22 in total, Veh=6, aldosterone treated samples=8, fluticasone treated samples=8).* p<0.05 vs vehicle group, ANOVA; # p=0.024 vs vehicle group, Mann Whitney U test. Boxes show median and interquartile range, with whiskers from minimum to maximum. In addition, the mRNA levels of Bcan (P=0.071, F(1, 10)=4.20), Vcan (P=0.985, F(1, 10)=0.00), Hapln4 (P=0.528, F(1, 10)=0.43) and Gad1 (P=0.434, F(1, 10)=0.67) were not affected by collagen 3 at 14 DIV, suggesting that mRNA levels of Bcan , Vcan, Hapln4 and Gad1 were not affected through an action of glucocorticoids binding to GPR56/97 ( Fig. 8 B ). In order to explore the mechanisms involved in these non-GR actions further, we compared the effects of the selective MR agonist aldosterone with the selective (no MR actions) GR agonist fluticasone. The mRNA expression of Bcan (P=0.938; F(2,22)=1.77), Vcan (P=0.440; F(2,22)=1.02), Hapln4 (P=0.887; F(2,22)=3.30) and Gad1 (P=0.0.981; F(2,22)=0.68) remained unchanged after exposure to aldosterone at 14DIV for 4h ( Fig 8 C ). In terms of the effect of fluticasone, there was a decreasing tendency of Hapln4 mRNA expression (P=0.060; F(2,22)=1.77; Veh vs fluticasone, p=0.024, Mann Whitney U test) ( Fig 8 C ), suggesting an inhibition effect of fluticasone mediated by GRs. However, there was no significant change for Bcan (p=0.201; F(2,22)=1.77), Vcan (p=0.460; F(2,22)=1.02) or Gad1 (p=0.636; F(2,22)=0.68) expression ( Fig 8 C ). These results indicated that MRs were almost certainly not the mediator of the effects of CORT on the expression of these genes. Further, the clear lack of effect of fluticasone in the cases of Vcan and Gad1 provided additional evidence that GRs are also not involved in these effects. No evidence for GR-mediated mRNA decay involvement The GRs in the cytoplasm reportedly can bind directly to RNA, and GCs can activate the RNA-bound GR, leading to mRNA degradation, defined as the GC-mediated mRNA decay (GMD) pathway (Cho et al., 2015). However, no alteration in the rate of Bcan or Hapln4 mRNA degradation was detected after exposure to 20nM CORT (Supplementary figure S9). Glucocorticoids suppress proteasome activity To test whether the glucocorticoids affect proteasome activities, neuronal cultures at 14DIV, treated with hydrocortisone for 4h, were measured with fluorogenic proteasome assay with 3 different proteasome substrates related to the 3 proteasome activities, including chymotrypsin-like activity, caspase-like activity, and trypsin-like activity. All activity was completely inhibited by 10μM MG132 (data not shown). The results of the proteasome assays showed that low and high doses of CORT tended to inhibit the proteasome activities. The chymotrypsin-like activity could be inhibited by both low dose and high dose CORT compared to the vehicle group ( Fig. 9 ), however, only the inhibition by high dose CORT was statistically significant (F (2, 11) =7.18, p=0.011 veh vs high dose CORT; p=0.144 veh vs low dose CORT, post-hoc Tukey test). However, no apparent effect of low or high dose CORT on caspase-like activity was observed (F (2, 11) =0.32, p=0.910 veh vs low dose CORT; p=0.923 veh vs high dose CORT, post-hoc Tukey test) ( Fig 9 ). Moreover, only very low activity was observed using the substrate which detected the trypsin-like activity (data not shown). Although the inhibition tendency was shown with high dose CORT exposure, the inhibition was not significant (F (2, 11) =2.65, p=0.947 veh vs low dose CORT; p=0.219 veh vs high dose CORT, post-hoc Tukey test). Download figure Open in new tab Figure 9: 20S proteasome activities (chymotrypsin-like, caspase-like and trypsin-like activity from cortical cultures at 14DIV treated with veh (distilled water), 20nM or 100nM CORT. n=4/group. *p<0.05 vs vehicle group (post-hoc Tukey test). Results are shown as mean +/− s.e.m. with individual data points. While PNNs are not detectable at 7 DIV, the widespread and diverse suppressive effects of GCs on the genes encoding components of PNNs that we have observed at 14 DIV strongly suggest that GC exposure is likely to have detrimental effects on PNN formation at this stage, but potentially not at 21 DIV. To clarify the impact of glucocorticoids on dendritic and structural formation of PNNs, WFA-labelling was monitored in cultured cells exposed to the same treatments. At 14 DIV, exposure to 20nM or 100nM CORT resulted in decreased length of PNNs covering dendrites after 4h (F (2, 946) =56.11, p<0.001) (data not shown), with reduction of mean length from 29.3µm to 23.4µm with 20nM CORT (Tukey post hoc tests, veh vs 20nM CORT, p<0.001) and to 21.7µm with 100nM CORT exposure (Tukey post hoc tests, veh vs 100nM CORT, p<0.001). Length of dendrites covered by PNNs also decreased after 24h (F (2, 673) =14.89, p<0.001) CORT exposure ( Fig.10 A,B ), with reduction of mean length from 25.3µm to 21.0µm with 20nM CORT (Tukey post hoc tests, p<0.001) and to 23.4µm with 100nM CORT exposure (Tukey post hoc test, p=0.044). Similarly, the brightness intensity of WFA-labelled PNNs covering dendrites also decreased after 24h (F (2, 673) =14.89, p<0.001) with both 20nM and 100nM CORT treatment, from 23.09 a.u. to 20.13 a.u. and 16.9 a.u., respectively (p=0.024 Veh vs 20nM Tukey post hoc test; p<0.001, Veh vs high dose, Tukey post hoc test) ( Fig.10 A,C ); however, no significant changes in brightness of staining were found after 4h (F (2, 946 =1.12, p=0.327) treatment (data not shown). Download figure Open in new tab Figure 10. Effect of 24h glucocorticoid exposure on WFA-labelled PNNs in cultured neurons at 14 or 21 DIV A : representative images of WFA-labelled PNNs around cultured neurons at 14 DIV after 24h exposure to vehicle, 20nM or 100nM CORT. B : length of dendrite covered by PNN, and C : brightness intensity of WFA staining (Veh: 270 dendrites in 20 cells nested in 3 different slides with 2 cultures, 20nM: 166 dendrites in 20 cells nested in 3 different slides with 2 cultures, 100nM: 199 dendrites in 16 cells nested in 3 different slides with 2 cultures). D : representative images of WFA-labelled PNNs around cultured neurons at 14 DIV after 24h exposure to vehicle, 20nM or 100nM CORT in the presence of mifepristone (20nM). E : length of dendrite covered by PNN, and F : brightness intensity of WFA staining (Veh: veh: 139 dendrites in 13 cells nested in 3 different slides with 2 cultures, 20nM: 275 dendrites in 17 cells nested in 3 different slides with 2 cultures, 100nM: 164 dendrites in 15 cells nested in 3 different slides with 2 cultures; Mif: veh: 290 dendrites in 21 cells nested in 3 different slides with 2 cultures, 20nM: 182 dendrites in 16 cells nested in 3 different slides with 2 cultures, 100nM: 177 dendrites in 14 cells nested in 3 different slides with 2 cultures). G,H: Effects on PNNs at 21 DIV after 24 treatment – G: length of dendrite covered by PNN, and H : brightness intensity of WFA staining (Veh: veh: 360 dendrites in 32 cells nested in 3 different slides with 2 cultures, 20nM: 263 dendrites in 20 cells nested in 3 different slides with 2 cultures, 100nM: 265 dendrites in 20 cells nested in 3 different slides with 2 cultures; Mif: veh: 165 dendrites in 20 cells nested in 3 different slides with 2 cultures, 20nM: 297 dendrites in 18 cells nested in 3 different slides with 2 cultures, 100nM: 291 dendrites in 19 cells nested in 3 different slides with 2 cultures). ### p<0.001 ANOVA main effect mifepristone vs vehicle group ** p<0.01, *** p<0.001 post-hoc Tukey test vs corresponding vehicle control group. Scale bars represent 20μm. Boxes show median and interquartile range, with whiskers from minimum to maximum. Moreover, at 21DIV, there was still an overall effect of CORT and of mifepristone on the length of PNN-covered dendrites after 24h treatment (F (1, 1580)) = 47.13, p<0.001). The interaction of CORT treatment and mifepristone was not quite significant (F (2, 1580)) = 2.81, p=0.060), but with a decreasing tendency in mean length of PNNs covering dendrites from 22.5µm to 19.0µm after 24h 100nM CORT with co-treated mifepristone (Tukey post hoc tests, p<0.001). However, no overall effect of mifepristone on the brightness intensity of WFA-labelled PNNs covered dendrites was found after 24h CORT and mifepristone treatment (F (1, 1580)) = 47.13, p=0.576) ( fig.4 C D), however, there was a marginally significant interaction of CORT and mifepristone treatment (F (2, 1580)) = 3.06, p=0.047) with slightly increased brightness intensity of WFA-labelled PNNs after 100nM CORT and mifepristone treatment (Tukey post hoc test, p=0.046). Hence we observe robust decreases in the length of proximal dendrite covered by PNN after CORT exposure, at both 14 and 21 DIV. These effects on PNN structure do not appear to be mediated via GRs. Since loss of TnR reportedly reduces the number of dendrites that are covered proximally by PNNs ( Weber et al., 1999 ), we also checked this parameter. However, no change in the number of WFA-labelled dendrites/cell was observed after CORT or mifepristone exposure (Supplementary figure S10). Discussion A strength of this study is that the expression of all the genes encoding core PNN components has been monitored in parallel. Hence the relative effects on the different genes can be interpreted with confidence. Our initial expectation was that we might identify one or two components of the PNN where gene expression is modulated by CORT levels. Instead, we found a widespread and complex regulation of multiple PNN component genes, via diverse mechanisms, and suggesting a prominent role for GCs in modulating PNN gene expression, but potentially limited to early in development. The diminishing evidence for GC regulation of these genes from 7 to 21 DIV suggests that these gene expression mechanisms are very important while PNNs are forming and stabilising, but are much less important once they have attained a mature configuration. Only for the phosphacan/ Ptprz1 gene was there a complete lack of evidence for regulation by GCs. Even in this case, GC modulation may be just hidden rather than absent. The exons encoding the phosphacan protein are also present in longer mRNAs encoding the full length Ptprz1 protein, and so any regulation specific for the phosphacan isoform might not be evident among the total transcripts, although a genomic action should still be evident, as, occurring prior to mRNA splicing, it should affect all protein isoforms. Mechanisms of GC regulation of PNN gene expression Basal human serum cortisol concentrations are in the range of 150 to 350 nM, although around 80% is not free in solution, but is bound to cortisol-binding globulin ( Hägg et al., 1987 ; Khalili-Mahani et al., 2015 ; Oster et al., 2016 ; Taylor et al., 1983 ), leaving free concentrations of around 30-70nM, although there is also marked circadian oscillation. Under conditions of high psychological stress, levels can rise to over 1000nM ( Chatterton et al., 1997 ; Kotozaki & Kawashima, 2012 ; Vanhorebeek et al., 2006 ). Similarly, in mice, resting total corticosterone levels are around 100-350nM (free concentrations ∼ 20-70nM), rising to 1000-3000nM (free concentrations ∼ 200-600nM) during stress ( Gong et al., 2015 ). During pregnancy, where mothers are not stressed, foetal cortisol levels have been estimated to be around 55nM ( Gitau et al., 1998 ). Neuronal survival in culture relies on the presence of basal levels of GCs. Neurobasal medium with B27 supplement (which does not contain cortisol binding globulin) contains CORT concentrations of around 50-70nM ( Brewer et al., 1993 ; Chen et al., 2008 ; Crochemore et al., 2005 ; Roth et al., 2010 ; Sünwoldt et al., 2017 ), in fact very similar to basal free GC concentrations in a non-stressed condition. Accordingly, we selected 2 concentrations of CORT to be added as experimental manipulation in this study, to represent both physiological (20nM) and pathological (100nM) levels of stress response. Resting GC concentrations will be fully activating MRs, whereas increasing concentrations under stress will additionally stimulate increasing proportions of GRs (and both the 20nM and 100nM concentrations will also activate GPR56/97 ( Barros-Álvarez et al., 2022 ; Cain et al., 2023 ; de Kloet, 2022 ; Y. Q. Ping et al., 2021 ; Rafestin-Oblin et al., 1986 ). In fact, where we observed effects of 100nM CORT, we generally also observed the effect with the lower concentration. At 7DIV, these effects were mostly mediated by GRs, as indicated by sensitivity to mifepristone. Where a significant effect was detected with the higher but not the lower concentration (as in suppression of Gad1 and Has3 expression), the same trend was observed with the lower concentration, consistent with dose-dependency at a single receptor. These effects at 7DIV, affecting many PNN component genes, reveal a widespread sensitivity of PNN component gene expression to GC levels at this developmental time, mediated by GRs. At 7DIV, CORT suppressed Ncan and TnR expression, and mifepristone enhanced Acan expression and further suppressed Ncan and TnR expression. Thus basal GC levels are tending to promote Ncan and TnR expression relative to Acan expression, and there are additional non-GR-mediated GC actions to dampen down Ncan and TnR expression. Modulation of Has gene expression is prominent at this developmental stage - CORT suppressed Has 1,2 and 3 expression via GRs, whereas at 14DIV, this effect was not evident, but mifepristone elevated Has 1 and 2 expression. This implies that GCs are still acting to reduce Has 1 and 2 expression through GR activation at 14DIV, but that the effect has become more sensitive, so that the low GC concentrations in the culture medium are now effective. A powerful GC/GR-mediated suppression of Has 1,2 and 3 mRNA has been noted in peripheral cells ( McRae et al., 2017 ; Stuhlmeier & Pollaschek, 2004 ; Zhang et al., 2000 ). Apart from the elevated levels of Has1,2 mRNAs induced by mifepristone at 14 DIV, decreased Bcan expression was also observed, despite the lack of GC modulation at 7 DIV, and suggesting heightened mRNA levels at 14 DIV due to basal GC levels. The other effect detected at 14DIV was a pronounced suppression of Vcan and Hapln4 expression by CORT, where there also appeared to be increasing sensitivity to GCs, as no significant suppression was detected at 7 DIV, although, for the effect on Vcan mRNA levels at 14 DIV, GRs appeared not to be involved. Conversely, mRNA levels for Ncan, TnR and Gad1 had now become insensitive to GCs. While reduced expression of Bcan, Ncan and Vcan mRNA expression following GC exposure appears not to have been previously reported in the CNS, both the Acan and Ncan gene promoters contain GR-response elements ( Rauch et al., 1995 ; Watanabe et al., 1995 ), and GC suppression of Vcan and Acan expression has been noted in peripheral cells ( McRae et al., 2017 ; Short et al., 2020 ; Song et al., 2012 ). Interestingly, prenatal exposure to GCs downregulates peripheral tissue Acan expression ( Chen et al., 2018 ), suggested that early developmental exposure can have lasting effects in offspring. There is some existing evidence suggesting suppression of Ncan expression by stress or GC exposure. Liu et al. ( Liu et al., 2008 ) reported GC-induced downregulation of expression of Ncan in astrocyte cultures (1 DIV) over 48h, partially mifepristone-sensitive, and intracerebral dexamethasone also reportedly decreased Ncan immunoreactivity ( Zhong & Bellamkonda, 2007 ). Chronic stress in adolescence or in adulthood in rodents also seems to suppress cortical or hippocampal Ncan expression ( Koskinen et al., 2020 ; Yu et al., 2022 ). Adult rodents exposed to stress show down-regulation at the protein level of hippocampal Bcan, Ncan, Ptprz1, TnR and Hapln1 but not Acan ( Koskinen et al., 2020 ) and PFC Acan but not Bcan ( Li et al., 2024 ). The suppression of Ncan expression by GCs at 7 DIV was not attenuated by mifepristone, but a similar suppression was detected following exposure to collagen 3, an agonist at Gpr56 and Gpr97. It is interesting to note that Ncan appears to be synthesised primarily by astrocytes and glutamatergic projection neurons ( Huntley et al., 2020 ; Irala et al., 2024 ; Zhang et al., 2014 ) (there will be a small proportion of astrocytes present in our neuronal-enriched cultures), and Gpr56 is expressed predominantly in astrocytes (but not glutamatergic projection neurons ( Chiou et al., 2021 ; Huntley et al., 2020 ; Zhang et al., 2014 ). Hence it is possible that GCs can act via GRs to produce a generalised enhancement of Ncan expression, whereas GC activation of Gpr56 can reduce Ncan expression specifically in astrocytes. GCs are reported to exert a post-transcriptional regulation of gene expression by GC/GR complexes affecting mRNA stability (Cho et al., 2015). However, we did not obtain any evidence to support this mechanism of action for the regulation of PNN component gene expression. Indeed, for many GC-induced changes in PNN component (suppression of Bcan, Vcan and TnR mRNA levels), we were unable to pinpoint the mechanisms of GC action. GRs appeared not to be involved (since mifepristone had little ability to attenuate the effects), and neither did Gpr56/97 (as reflected in the inability of collagen 3 to replicate the effect), but the effects seemed rapid, in that they were detectable within 4h, and so are likely to be non-genomic in nature. Effect on PNN structure Compromised PNN formation (brightness of WFA staining) in cortex and hippocampus after exposure to prenatal or neonatal stress is well-documented in rats and mice ( Allgäuer et al., 2023 ; Gildawie et al., 2020 ; Jakovljevic et al., 2022 ; Riga et al., 2017 ; Santiago et al., 2018 ). Most studies using mouse hippocampal or cortical cultures concur that at 14 DIV, PNNs are almost mature, with a net-like structure covering somata and proximal dendrites, and from then on the morphology changes little ( Dityatev et al., 2007 ; Fowke et al., 2017 ; Geissler et al., 2013 ). Our observations were similar. This report may be the first demonstration that GCs modify PNN structure directly, and hence may be the mediators of the effects of stress on PNNs. GC exposure caused a small but robust reduction in the length of PNN ensheathing the proximal dendrites, not involving GRs, and a more slowly-developing increase in intensity of WFA staining, likely to be mediated by GRs. The different mechanisms involved suggests that the increased brightness of WFA staining is not simply a result of compaction of a constant amount of PNN into a smaller volume. At 14 DIV, the magnitude of the changes in PNN structure might be considered smaller than expected, considering the quite profound changes in gene expression observed with GC exposure. However, PNNs seem to be quite robust to altered expression of component genes. While loss of Acan, phosphacan, TnR or Hapln4 compromises PNN structure ( Bekku et al., 2012 ; Brückner et al., 2000 ; Eill et al., 2020 ; Giamanco et al., 2010 ; Haunsø et al., 2000 ; Morawski et al., 2014 ; Rowlands et al., 2018 ; Sucha et al., 2020 ; Suttkus et al., 2014 ; Weber et al., 1999 ), PNNs appear unaffected even in the complete absence of Bcan, Ncan or Has3 ( Arranz et al., 2014 ; Brakebusch et al., 2002 ; Zhou et al., 2001 ). The suppression of Bcan , Vcan and Hapln4 gene expression by GCs at 14 DIV was not mifepristone-sensitive, and equally the decrement in PNN structure was also not mifepristone-sensitive. However it seems unlikely that the altered WFA staining is due to alterations in PNN component gene expression. Changes in PNN component gene expression detected when HCA was applied at 14 DIV were not attenuated by mifepristone, whereas alterations in WFA staining characteristics at 14 DIV were sensitive to mifepristone. Equally, no changes in PNN component gene expression were detected from GC exposure at 21 DIV, yet altered WFA staining characteristics are still observed. We have not systematically profiled PNN component protein expression at 21 DIV, so post-transcriptional actions of GCs may be responsible. For example, it has been suggested that Acan levels are partly controlled by post-translational modifications (Zhu, Cui, et al., 2019), so if these processes were modulated by GCs, a subtle but rapid effect on PNN structure might be evident. Modulation of GABAergic interneuron genes Expression of both Has3 and Hapln4 (suppressed by GCs at 7 and 14 DIV) appears to be markedly enriched in Pvalb+ve cells as compared to other cortical neurons ( Huntley et al., 2020 ). The regulation of Has3 and Hapln4 mRNA levels (and protein levels for Has3) by GCs might suggest a particular action of GCs on Pvalb+ve cells. Elevated GC levels suppressed Gad1 expression (7 and 14 DIV) and Pvalb expression (21 DIV), and at 7 DIV, blockade of GRs with mifepristone elevated expression of both Gad1 and Pvalb . These 2 GABAergic interneuron genes, both of which show reduced expression levels in schizophrenia, are clearly sensitive to elevated GC levels. The action on Gad1 expression at 7 DIV and 14 DIV appeared to involve GRs, whereas the action on Pvalb expression at 21 DIV, when PNN component genes were apparently resistant to GC actions, involved another mechanism. These results are consistent with some previous observations. There are reports that chronic stress prenatally ( Allgäuer et al., 2023 ; Heslin & Coutellier, 2018 ; Uchida et al., 2014 ; van de Looij et al., 2019 ), neonatally ( Murthy et al., 2019 ) and in adult rodents ( Banasr et al., 2017 ; Hu et al., 2010 ) decreases cortical Pvalb expression, although increased expression after adult stress has also been reported ( Page et al., 2019 ). High GC concentrations for 72h also reportedly suppress Gad1 (but not Gad2 ) expression in 10 DIV cultured cortical neurons ( Banasr et al., 2017 ), and prenatal GC exposure in vivo reduces offspring Pvalb expression in hippocampus ( Zhang et al., 2023 ). Hence our data, along with previous reports, seems clear in identifying Pvalb+ve cells potentially as direct targets of GCs for inducing gene expression changes relevant to schizophrenia. Proteasome Proteasome inhibition leads to elevated cellular levels of proteins which are proteasome substrates. The modulation of neuronal proteasome activity by GCs has not previously been reported. There are some hints of proteasome modulation by GCs from peripheral cells, although for stimulation rather than inhibition. In hepatocytes, dexamethasone stimulates protein degradation within 4h (Hopgood et al, 1980), and similarly in muscle cells GCs accelerate protein degradation via ubiquitin pathways ( Sun et al., 2008 ). There is a single report, in thymocytes, of physiologically-relevant concentrations of dexamethasone suppressing chymotrypsin-like and caspase-like, but not trypsin-like, proteasome activity within 3h ( Beyette et al., 1998 ). Here we report an inhibition of neuronal proteasome activity that is specific for chymotrypsin-like activity as compared to caspase-like (post-glutamate peptide hydrolase-like) activity (trypsin-like activity was very low, and it might be more difficult to observe suppression of activity). However, it should be noted that this suppression of chymotrypsin-like activity by CORT was demonstrated at 14DIV, while at this stage CORT decreased HAS2 protein levels, rather than the increase detected at 7DIV. It seems likely that a similar action of glucocorticoids on the proteasome occurs at 7DIV, but additional experiments would be needed to formally demonstrate this, potentially implicating suppression of proteasome activity in the Has2 protein increase. Has2 protein has an extremely short half-life (∼15minutes), and there is some evidence suggesting that its (lack of) longevity might be controlled by the proteasome at least in peripheral cells ( Karousou et al., 2010 ; Vigetti et al., 2012 ), so elevated protein levels 4h after exposure to GCs are consistent with reduced degradation via proteasome inhibition. Conclusions Perinatal trauma, which increases offspring schizophrenia risk ( Davies et al., 2020 ; Paquin et al., 2021 ), elevates neonatal cortisol levels ( Chiș et al., 2017 ; Gardner et al., 2001 ; Hernandez-Andrade et al., 2005 ; Improda et al., 2023 ). At this period, corresponding to our 7-14 DIV cultured neurons in mice, as PNNs form and stabilise, cortical Ncan and Vcan expression is declining, while TnR and Pvalb expression is increasing, in mice ( Brückner et al., 2000 ; Du et al., 1996 ; Fertuzinhos et al., 2014 ; Fuss et al., 1993 ; Milev et al., 1998 ; Nowicka et al., 2009 ), and in humans ( Honig et al., 1996 ; Kang et al., 2011 ; Letinic & Kostovic, 1998 ; Rogers et al., 2018 ). Our data suggest that inappropriately elevated cortisol levels at this time will be sufficient to disrupt the expression of PNN component genes at a critical time for PNN formation. 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Share Glucocorticoids modulate expression of perineuronal net component genes and parvalbumin during development of mouse cortical neurons Liang Yue , Michael T Craig , Brian J Morris bioRxiv 2025.02.12.637910; doi: https://doi.org/10.1101/2025.02.12.637910 Share This Article: Copy Citation Tools Glucocorticoids modulate expression of perineuronal net component genes and parvalbumin during development of mouse cortical neurons Liang Yue , Michael T Craig , Brian J Morris bioRxiv 2025.02.12.637910; doi: https://doi.org/10.1101/2025.02.12.637910 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18589) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)