Psychosocial stress and cannabinoid drugs affect acetylation of α-tubulin (K40) and gene expression in the prefrontal cortex of adult mice
Psychosocial stress, cannabinoid drugs and acetylation of α-tubulin
Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Göttingen, Germany
Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University of Göttingen, Göttingen, Germany
Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology–Developmental Biology, GZMB, Georg-August-University Göttingen, Göttingen, Germany
University of Maryland at College Park, UNITED STATES
* E-mail: jordi.tomas-roig@med.uni-goettingen.de (JTR); shoyer@gwdg.de (SHF)Abstract
The dynamics of neuronal microtubules are essential for brain plasticity. Vesicular transport and synaptic transmission, additionally, requires acetylation of α-tubulin, and aberrant tubulin acetylation and neurobiological deficits are associated. Prolonged exposure to a stressor or consumption of drugs of abuse, like marihuana, lead to neurological changes and psychotic disorders. Here, we studied the effect of psychosocial stress and the administration of cannabinoid receptor type 1 drugs on α-tubulin acetylation in different brain regions of mice. We found significantly decreased tubulin acetylation in the prefrontal cortex in stressed mice. The impact of cannabinoid drugs on stress-induced microtubule disturbance was investigated by administration of the cannabinoid receptor agonist WIN55,212–2 and/or antagonist rimonabant. In both, control and stressed mice, the administration of WIN55,212–2 slightly increased the tubulin acetylation in the prefrontal cortex whereas administration of rimonabant acted antagonistically indicating a cannabinoid receptor type 1 mediated effect. The analysis of gene expression in the prefrontal cortex showed a consistent expression of ApoE attributable to either psychosocial stress or administration of the cannabinoid agonist. Additionally, ApoE expression inversely correlated with acetylated tubulin levels when comparing controls and stressed mice treated with WIN55,212–2 whereas rimonabant treatment showed the opposite.
Data Availability
All relevant data are within the manuscript and its Supporting Information files.
Introduction
Environmental factors such as traumatic events and consumption of drugs of abuse like cannabinoids can induce psychotic disorders as a result of neurobiological changes. These alterations comprise dysfunctions in neurogenesis, axonal growth, myelinization, synaptogenesis, synaptic pruning, and neuroendocrine regulation [1]. For instance, preclinical studies indicated that chronic exposure to psychosocial stress can cause neuroanatomical changes as e.g. inhibition of adult neurogenesis in the dentate gyrus, or dendritic atrophy in the hippocampus (HIPP) and the prefrontal cortex (PFC) [2–5]. Furthermore, chronic exposure to stress compromised the stability and function of axonal microtubules (MTs) promoting the hyperphosphorylation of the microtubule-binding protein Tau, and the formation of neurofibrillary tangles [6, 7]. Cytoskeletal alterations have also been reported in several neuropsychiatric diseases such as schizophrenia, major depressive disorder, and bipolar disorder. A common hallmark underlying neuropsychiatric disorders comprises an aberrant expression of tubulin isoforms and tubulin acetylation [8, 9].
MTs are assembled from stable heterodimers of α/β-tubulin and are constantly remodeled. In neuronal cells, MTs are important for compartmentation, rigidity, long-distance transport, and synaptic transmission [10]. Although they are highly conserved in evolution, MTs adapt to diverse cellular functions through microtubule-associated proteins (MAPs), posttranslational modifications of tubulins (PTMs), and binding proteins for specific PTMs. Thus, posttranslational modifications generate a ‘tubulin code’ that specifies the assignment and coordination of the complex functions of MTs [11–13]. An important modification is the acetylation of α-tubulin at lysine 40 (α-tubulin K40ac) that has been generally associated with MT stability. The addition of acetyl groups does not alter the MT ultrastructure but it is required for vesicular transport [14, 15]. The major tubulin acetyltransferase in mammals is the α-tubulin acetyltransferase 1 (ATAT1) causing complete loss of tubulin acetylation when deleted in mice. Although the loss of ATAT1 is not life-threatening, rodents underwent an enlargement of the forebrain lateral ventricles pointing out an important biological function of the Atat1-gene in this brain area [16–18]. Deacetylation of α-tubulin at K40ac is achieved by the histone deacetylase 6 (HDAC6) and the sirtuin type 2 (SIRT2), which, additionally, deacetylate further substrates [19, 20]. In the rodent brain, the main α-tubulin deacetylase is HDAC6 [21]. Additionally, HDAC6 recruits polyubiquitinated proteins to aggresomes and controls the fusion of autophagosomes and lysosomes [22–24]. HDAC6 is, therefore, involved in the clearance of misfolded proteins and protein aggregates [25, 26]. Furthermore, HDAC6 activity has been associated with emotional behavior like activity, anxiety, and depression [27, 28]. Thus, MTs in general and HDAC6, in particular, came into focus as promising therapeutic targets for neuropsychiatric disorders [29, 30]. To this end, HDAC inhibitors are emerging as potential antidepressant drugs [31].
Under physiological conditions, synaptic transmission and tissue homeostasis are biological mechanisms regulated by the cannabinoid receptor type-1 through G protein-coupled receptors (GPCRs) [32]. The cannabinoid receptor type-1 (CB1) is highly expressed in the brain and, specifically, found in axons and presynaptic terminals [33]. Upon stress exposure, CB1 and its endogenous ligands compromise the proper function of the brain [34]. CB1 mediates the pharmacological actions of synthetic cannabinoid drugs such as the full cannabinoid agonist WIN55,212–2 (referred to as W) and the inverse agonist rimonabant (referred to as R) [35]. Activation of CB1 by endogenous or exogenous cannabinoids regulates neuronal metabolism by decreasing mitochondrial cAMP and PKA [36]. Under pathological conditions of prolonged exposure to a stressor, the expression of CB1 is compromised [32, 35]. Socially defeat mice display a variety of molecular and physiological changes, commonly reported in psychiatric disorders, including changes in the endocannabinoid system and deregulation of β-actin contributing to dendritic and synaptic dysfunctions [7, 37, 38].
Here, we used a long-term psychosocial defeat protocol as a stress model for its etiological, predictive, discriminative, and face validity [39]. In our model, mice were exposed to daily psychosocial stress and finally acutely treated with the full cannabinoid agonist W and/or the inverse agonist R. We hypothesized that MTs might be affected in stressed animals, and thus, treatment with cannabinoid drugs could confer neuroprotection by remodeling the MT system. For this purpose, we evaluated stressed animals treated with cannabinoid drugs by quantifying α-tubulin acetylation and assessing differential gene expression. Among the wide spectrum of cerebral regions closely involved in stress-related disorders (for review see Ref. [40]), we directed our investigation on the PFC, a brain structure critically involved in social behaviour [41, 42].
Materials and methods
Ethics statement
All procedures were approved by the Göttingen University Institutional Animal Care and Use Committee and were in accordance with NIH guidelines for the use of animals in research and the European Communities Council Directive (2010/63/EU). The study was designed and carried out in compliance with the ARRIVE guidelines.
Drug treatments
The CB1 receptor agonist WIN55,212–2 (referred to in the text as W, Sigma-Aldrich, St. Louis, USA) and the selective cannabinoid CB1 inverse agonist rimonabant (referred to in the text as R, Sequoia Research Products Ltd., Pangbourne, UK) were dissolved in a vehicle solution consisting of 10% DMSO (Sigma-Aldrich, St. Louis, USA) and 0.1% Tween-80 (Sigma-Aldrich, St. Louis, USA) in 0.9% saline. On day 21 of the experiment, the animals were injected intraperitoneally with a volume of 200 μl of drug and/or vehicle and then evaluated by behavioral testing [34]. The drugs W and R were administered at a concentration of 3mg/kg. Control and stressed animals were further divided into four subgroups of fifteen mice each, which received different treatments. Mice were treated twice with vehicle (V+V) as the control group, or subjected first to the vehicle and then W (V+W), or treated first with R and then with W (R+W), or injected first with R and then with vehicle (R+V).
Sample preparation
Animals were sacrificed immediately after finishing the experiment. All mice were deeply anesthetized by intraperitoneal injection of 2,2,2-tribromethanol (Sigma-Aldrich, St. Louis, USA) followed by transcardial perfusion with cold 0.1% phosphate-buffered saline (PBS). Brain samples were isolated and frozen in liquid nitrogen. For the Western blot analyses, the brain samples from four randomly selected animals from each subgroup were chosen, giving a total of 32 animals. On the day of testing, tissues were homogenized in RIPA buffer containing protease inhibitors (Roche Applied Science, Penzberg, Germany). SDS-containing reducing sample buffer was added giving a final concentration of 1x [43]. Probes were denatured for 5 min at 60°C.
Immuno-blotting
Protein lysates were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Hybond-ECL, GE Healthcare) [43, 44]. The membrane was blocked in 5% dry milk in TBST (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween20) for one hour followed by incubation with the primary antibodies. These were mouse anti-acetylated α-tubulin (clone 6-11B-1; Santa Cruz, #sc-23950), and rabbit anti-ß-actin (Proteintech, #20536-1-AP). Both antibodies were resuspended in blocking solution and incubated with the blots at 4°C overnight by constant agitation in roller tubes. Blots were washed for 30 minutes in TBST followed by incubation with fluorophore-labelled secondary antibodies goat anti-mouse IRDye800CW (LI-COR Biosciences, #925–32210, lot #C81106-01), and goat anti-rabbit IRDye680RD (LI-COR Biosciences, #925–68071, lot #C80911-11) for 45 minutes at room temperature in roller tubes. After 45 minutes of washing in TBST, fluorescent images were captured by Odyssey CLx Imaging System (LI-COR Biosciences, Nebraska, USA), and proteins were quantified using the software Image StudioTMLite (LI-COR Biosciences, Nebraska, USA).
Protein quantification
For each individual Western blot experiment, all protein samples from one brain region, including the four different biological replicates for each treatment, were loaded onto four separate gels and immune-blotting and image capture were performed concurrently, using identical antibody dilutions, and identical image capture settings. The amounts of acetylated tubulin and ß-actin were measured in each lane, and the relative amount of acetylated tubulin in each lane was determined by calculating the ratio of acetylated tubulin to ß-actin in the same lane. The fold change of relative tubulin acetylation was determined using the average of the relative tubulin acetylation of the four biological control probes (obtained from mice not subjected to psychosocial stress, and injected with vehicle only, CTR (V+V)) as the reference, which was set as 1. The fold changes were calculated for all probes investigated concurrently on a set of four blots. These immune-blotting experiments and their analyses were repeated up to five times, always loading all probes onto a set of four gels as described above. Each run of Western blots was analyzed separately. Thus, up to five technical repetitions were used for each biological replicate. The average values of the relative amount of acetylated-α-tubulin of all technical replicates for each of the biological replicates were calculated and used for statistical analyses. Four biological replicates, i.e. samples from four mice, were analysed for each treatment, except for the HIPP probes from stressed mice treated with W+V, for which protein lysates from only three biological replicates could be quantified.
Focused gene signature profiling
The prognostic 35-gene profile was performed in the digital transcript counting assay (nCounter-NanoString). The nCounter® technology permits the counting of individual nucleic acid molecules using digital detection of the fluorescent molecular barcodes attached to the target RNA. The mRNA hybridization, detection, and scanning were performed following the protocol provided by NanoString Technologies. 200–400 ng of RNA was taken as the starting material according to the manufacturer’s guidelines. Data were adjusted by scaling with the geometric mean of built-in control gene probes after log transformation (base 2) for each sample. The target genes were chosen according to the following criteria: genes involved in cytoskeleton architecture, neuropsychiatric disorders, and CNS myelination. The NanoStringnCounter™ code set was assigned as follows: β-actin (NM_007393.3), apolipoprotein E (ApoE; NM_009696.3), C-X-C motif chemokine 12 (CxCl12-Ɣ; NM_001012477.2), calreticulin (Calr; NM_007591.3), cannabinoid receptor 1 (Cnr1; NM_007726.3), cannabinoid receptor 2 (Cnr2; NM_009924.3), cub and sushi multiple domains 1 (Csmd1; NM_053171.2), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (Cnp; NM_001146318.1), diacylglycerol lipase (Dagl-α; NM_198114.2), discoidin domain receptor 1 (Ddr1; NM_001198831.1), dopamine receptor D1 (Drd1; NM_010076.3), dopamine receptor D2 (Drd2; NM_010077.2), dopamine receptor D3 (Drd3; NM_007877.1), dopamine receptor D4 (Drd4; NM_007878.2), dopamine receptor D5 (Drd5; NM_013503.2), dystrobrevin binding protein 1 (Dtnbp1; NM_025772.4), galactosylceramidase (Galc; NM_008079.3), glyceraldehyde-3-phosphate dehydrogenase (Gapdh; NM_008084.2), low density lipoprotein receptor (Ldlr; NM_001252658.1), myelin basic protein (Mbp; NM_001025251.2), myelin-associated glycoprotein (Mag; NM_010758.2), myelin oligodendrocyte glycoprotein (Mog; NM_010814.2), N-acyl phosphatidylethanolamine-specific phospholipase D (Nape-pld; NM_178728.5), nerve growth factor inducible (Vgf; NM_001039385.1), neuregulin 1 (Nrg1; NM_178591.2), oligodendrocyte transcription factor 1 (Olig1; NM_016968.4), oligodendrocyte transcription factor 2 (Olig2; NM_016967.2), reticulon 4 receptor (Rtn4r; NM_022982.2), retinoid X receptor alpha (Rxra; NM_011305.3), ryanodine receptor 3 (Ryr3; NM_177652.2), ski proto-oncogene (Ski; NM_011385.2), special AT-rich sequence-binding protein-2 (Satb2; NM_139146.2), SRY-box 10 (Sox10; NM_011437.1), zinc finger protein 488 (Zfp488; NM_001013777.2), and zinc finger protein GLI1 (Gli1; NM_010296.2). The mean value of the expression levels of Gapdh and β-actin was used as the standard control. Four mice per subgroup were used.
Statistical analysis
Pairwise comparisons between the controls and stressed animals and between specific drug treatment and the vehicle were evaluated by Student’s t-test (unpaired, two-tailed). For the t-test, the average values of the fold-change values of acetylated-α-tubulin of all technical replicates of each biological replicate were used (S1 Table). Excel was used to verify normal distribution of the data. A two-way ANOVA was used to determine the effects of long-term stress and drug treatment on the expression of acetylated tubulin. The mean differences among the levels of one factor were determined by one-way ANOVA. Pairwise comparisons were performed using Bonferroni post hoc test. Gene expression profile was determined as a two-tailed t-test on the log-transformed normalized data that assumed unequal variance using nSolver™ 4.0. The distribution of the t-statistic was calculated using the Welch-Satterthwaite equation for the degrees of freedom in the estimation of the 95% confidence limits for observed differential expression between the two groups. Statistics and graphs were shown using GraphPad Prism 9.1.0. The resulting p-values were adjusted according to Benjamini-Hochberg [45]. The significance was set at p ≤ 0.05. In all figures and text, data are represented as mean ± SE.
Results
Psychosocial stress affects tubulin acetylation in the brain
We have previously reported that daily psychosocial stress affects body weight, exacerbates scratching activity, and increases the frequency of urination [34]. We asked here, whether the cytoskeleton was affected by repeated psychosocial stress. To this end, we investigated α-tubulin acetylation at K40 in different brain regions of stressed (STS) mice and control (CTR) mice by quantitative immune-blotting. In each experimental setting four biological replicates, i.e., four individual male mice were investigated, and every probe was loaded several times. The protein lysates of all biological replicates were treated and analysed simultaneously. Acetylated α-tubulin (K40) and ß-actin were indirectly detected by fluorophore-labelled secondary antibodies, and the relative quantities of acetylated α-tubulin and their fold-change differences were calculated as described in the materials and methods section. Fold changes of all technical and biological replicates were combined in Fig 1 (CTR vs STS, all brain regions). We observed a decrease in α-tubulin acetylation in stressed animals compared to the control animals in the PFC, the HIPP, and the dorsal striatum (DS), whereas an opposite pattern was observed in the cerebellum (CRB). The changes in α-tubulin acetylation were statistically significant for the PFC (t(34) = 3.903; p<0.000485***). No significant differences were found for the DS (t(35) = 2.702; p = 0.2037), HIPP (t(37) = 1.696; p = 0.29900) and CRB (t(29) = 1.662; p = 0.12295). Subsequently, our data indicate that psychosocial stress affects the acetylation of α-tubulin in the PFC. Since the PFC is critically involved in social behavior [41, 42], we aimed to further characterize this brain structure by assessing which genes were differentially expressed when comparing stressed and control mice subjected to different pharmacological interventions.
Administration of the cannabinoid receptor agonist promotes tubulin acetylation in the prefrontal cortex
Since our results demonstrated an effect of psychosocial stress on acetylation of tubulin in the brain, which presumably influence the tubulin dynamics and the organisation of the cytoskeleton, its stability and function, we wondered whether cannabinoid treatments might be effective in overcoming the stress-induced impact on MTs and neural function. We focused here on the PFC due to its particular importance for social behaviour and intellectual abilities, along with the highly significant decrease of tubulin acetylation in stressed animals (Fig 1). The two way ANOVA revealed a significant effect of stress (F(1, 29) = 29.35; p<0.001***), drug treatment (F(3, 29) = 7.56; p = 0.001***) as well as a significant interaction (stress x drug) (F(3, 29) = 3.91; p = 0.022*). The interaction of stress and drug treatment was evaluated by multiple comparisons post hoc tests (Table 1). Bonferroni post hoc test revealed higher expression of acetylated tubulin in controls treated with either vehicle or the CB1 agonist when compared to their stressed counterparts (p = 0.0091**; p = 0.021*, respectively) (Table 1; Fig 2). The use of cannabinoid receptor agonist in controls increased the expression of acetylated tubulin in contrast to the control group treated with the antagonist (p = 0.014*) (Table 1; Fig 2) and also did so when both drugs were administered simultaneously under stress conditions in comparison with those subjected to stress and treated with the cannabinoid antagonist (p = 0.033*) (Table 1; Fig 2). Thus, our results indicate that treatment with the CB1 agonist protects from stress-induced tubulin deacetylation while the administration of the antagonist favours the removal of acetyl groups in the PFC.
| Equality of variances | One-way ANOVA | ||||||
| (Levene test) | |||||||
| Pairwise comparisons | (M i -M j ) + SD | p value | |||||
| Acetylated tubulin | W(7,22) = 0.99 | n.s. | F (7,22) = 8.84 | ***<0.001 | CTR V+V vs STS V+V | 0.41+0.01 | .009 |
| Expression | CTR V+W vs STS V+W | 0.43+0.11 | .021 | ||||
| CTR V+W vs CTR R+V | 0.42+0.10 | .014 | |||||
| STS R+V vs STS R+W | -0.35+0.09 | .033 | |||||
Acetylation of α-tubulin in the hippocampus, the dorsal striatum, and the cerebellum after administration of cannabinoid drugs
We also investigated α-tubulin acetylation in the HIPP, the DS, and the CRB after administration of either the cannabinoid receptor 1 agonist W, the inverse agonist R, or both cannabinoid drugs in both stressed and control animals. In the HIPP of control animals, administration of R+V caused a decrease in tubulin acetylation (t(26) = 3.577; p = 0.021577*) whereas neither V+W nor R+W resulted in a significant change in comparison to the control group treated with vehicle alone (Fig 3, panel A). In stressed animals, a significant change in tubulin acetylation was not observed compared to controls subjected to V+V, nor by drug administration. Administration of W+V in stressed animals caused a significant decrease in tubulin acetylation when compared with their matched controls (CTR, W+V, p = 0.020951*) (Fig 3, panel A).
In the DS, administration of either V+W, R+V, or R+W did not alter tubulin acetylation status in either the control or stress group. Significant changes in tubulin acetylation were observed in stressed animals treated with R+V or both drugs (R+W) when compared to their matched control group (p = 0.008861** and p = 0.034778*, respectively) (Fig 3, panel B).
In the CRB of control animals, the administration of either V+W, R+W, or R+V induced a significant decrease in tubulin acetylation as compared to the vehicle-treated controls (CTR, V+V vs V+W: t(24) = 3.102; p = 0.003811**; V+V vs R+W: t(23) = 2.896; p = 0.019340*; V+V vs R+V: t(23) = 3.793; p = 0.002640**) (Fig 3, panel C). Stressed animals treated with vehicle did not display significantly different tubulin acetylation than their matched controls. Furthermore, the administration of either V+W, R+V, or both cannabinoid drugs did not change tubulin acetylation in stressed animals compared to socially-defeat animals subjected to vehicle alone (Fig 3, panel C).
To conclude, the effect on tubulin acetylation by either long-term exposure to stress or administration of cannabinoid drugs was different according to the brain region but especially prominent in the PFC. The effects of stress are brain-region-specific as pointed out previously [46].
Gene expression signature in the prefrontal cortex
We reported changes in gene expression in the PFC when socially defeat mice were subjected to cannabinoid drugs by use of the digital transcript counting (nCounter) assay (NanoString) [35]. The target genes were chosen according to the following criteria: genes involved in cytoskeleton architecture, neuropsychiatric disorders, and CNS myelination. Long term exposure to psychosocial stress increased the expression of ApoE (t(6) = 6.61; padj = 0.01**), CxCl12ɣ (t(6) = 6.47; padj = 0.02*), Dtnbp1 (t(6) = 5.85; padj = 0.02*), Ski (t(6) = 5.16; padj = 0.04*), and Cnr1 (t(6) = 4.71; padj = 0.05*) when compared to their matched controls (Table 2). Non-stressed animals treated simultaneously with both drugs underwent a remarkable downregulation of ApoE (t(6) = -7.92; padj<0.001***) and Rtn4r (t(6) = -9.12; padj<0.001***) in comparison with their controls (Table 2). The administration of R+W in non-stressed mice resulted in lower levels of ApoE (t(6) = 11.07; padj<0.001***), Rtn4r (t(6) = 11.77; padj<0.001***), Vgf (t(6) = 7.4; padj = 0.01**), Rxra (t(6) = 6.92; padj = 0.01**), Cnp (t(6) = 5.6; padj = 0.02*), Mbp (t(6) = 4.72; padj = 0.05*), and Cnr1 (t(6) = 6.64; padj = 0.03*) than the control group treated with V+W (Table 2). Daily exposure to psychosocial stress induced a decrease in Mag expression upon V+W administration (t(6) = 5.73; padj = 0.04*) in contrast to those exposed to vehicle alone (Table 2). Social defeat mice treated with R+V displayed lower expression of ApoE (t(6) = -8.96; padj = 0.02*) than those treated with V+W (Table 2). Stressed mice treated with the cannabinoid agonist displayed an upregulation of ApoE (t(6) = 6.48; padj = 0.05*) when compared to their non-stressed counterparts (Table 2) Socially defeat mice subjected to R+W showed elevated levels of ApoE (t(6) = 9.18; padj<0.001***), Cnp (t(6) = 7.22; padj<0.001***), Vgf (t(6) = 8.35; padj<0.001***), Drd1 (t(6) = 7.81; padj<0.001***), Drd5 (t(6) = 5.95; padj = 0.02*), Rxra (t(6) = 7.2; padj<0.001***), Zfp488 (t(6) = 10.34; padj<0.001***), Mbp (t(6) = 6.92; padj = 0.01**), Cnr1 (t(6) = 6.51; padj = 0.02*), and Ryr3 (t(6) = 5.55; padj = 0.03*) in comparison to their counterparts non-exposed to stress (Table 2).
| Comparison | Gene symbol | Accession | Fold change | q-value |
|---|---|---|---|---|
| STS V+V vs CTR V+V | ApoE | NM_009696.3 | 1.44 | .01 |
| CxCl12ɣ | NM_001012477.2 | 1.54 | .02 | |
| Dtnbp1 | NM_025772.4 | 1.36 | .02 | |
| Ski | NM_011385.2 | 1.46 | .04 | |
| Cnr1 | NM_007726.3 | 1.63 | .05 | |
| CTR R+W vs CTR V+V | ApoE | NM_009696.3 | 0.55 | < .001 |
| Rtn4r | NM_022982.2 | 0.43 | < .001 | |
| CTR R+W vs CTR V+W | ApoE | NM_009696.3 | 0.45 | < .001 |
| Rtn4r | NM_022982.2 | 0.37 | < .001 | |
| Vgf | NM_001039385.1 | 0.46 | .01 | |
| Rxra | NM_011305.3 | 0.31 | .01 | |
| Cnp | NM_001146318.1 | 0.38 | .02 | |
| Cnr1 | NM_007726.3 | 0.49 | .03 | |
| Mbp | NM_001025251.2 | 0.32 | .05 | |
| STS V+W vs STS V+V | Mag | NM_010758.2 | 0.47 | .04 |
| STS R+V vs STS V+W | ApoE | NM_009696.3 | 0.80 | .02 |
| STS V+W vs CTR V+W | ApoE | NM_009696.3 | 1.40 | .05 |
| STS R+W vs CTR R+W | ApoE | NM_009696.3 | 2.23 | .001 |
| Cnp | NM_001146318.1 | 3.03 | .001 | |
| Vgf | NM_001039385.1 | 2.44 | .001 | |
| Drd1 | NM_010076.3 | 8.34 | .001 | |
| Rxra | NM_011305.3 | 3.28 | .001 | |
| Zfp488 | NM_001013777.2 | 2.90 | .001 | |
| Mbp | NM_001025251.2 | 3.90 | .01 | |
| Drd5 | NM_013503.2 | 4.18 | .02 | |
| Cnr1 | NM_007726.3 | 2.07 | .02 | |
| Ryr3 | NM_177652.2 | 4.76 | .03 |
Pearson’s chi-squared correlation analysis between gene expression and tubulin acetylation in the PFC
Administration of drugs had a consistent effect on tubulin acetylation in both control and stressed animals when the PFC was examined (Fig 4). The administration of the inverse agonist led to a decrease in tubulin acetylation when compared to the control group treated with vehicle whereas the administration of either R+V or R+W reduced tubulin acetylation in contrast to what was observed upon W treatment. We, therefore, analyzed the correlation between tubulin acetylation and gene expression signature reporting an inverse association between acetylated tubulin levels and either ApoE (r = -0.94; p<0.001***), CxCl12ɣ (r = -0.79; p = 0.019*), Dtnbp1 (r = -0.79; p = 0.019*), Ski (r = -0.98; p<0.001***) or Cnr1 (r = -0.87; p = 0.005**) expression when comparing controls and defeat mice subjected to vehicle (Fig 4, panel A); and also did so, after acute injection with V+W when compared the control and the stress group (r = -0.97; p = 0.001***) (Fig 4, panel B). A positive correlation between acetylated tubulin and ApoE levels was reported following either V+W or R+V under repeated stress (r = 0.74; p = 0.05*) (Fig 4, panel C).
Discussion
The dynamic rearrangement of the neuronal MTs is crucial for brain plasticity by enabling the remodelling of dendrites and axons [46]. Persistent stressful conditions revealed structural alterations of the cytoskeleton [6, 7]. We asked here, whether psychosocial stress affects the neuronal cytoskeleton by investigating the post-translational tubulin modification based on the addition of acetyl groups. We found a significant reduction of tubulin acetylation in the PFC, whereas non-significant differences were observed in the HIPP, the DS, and the CRB of mice exposed to chronic psychosocial stress. Reduced levels of tubulin acetylation in the PFC were accompanied by differential gene expression. Particularly, an upregulation of ApoE, CxCl12γ, Dtnbp1, Ski, and Cnr1 in the PFC of stressed mice was determined. The administration of V+W promoted an increase of both ApoE and Cnr1 levels in comparison to R+W. Upregulation of ApoE was also observed in stressed mice treated with V+W in contrast to their matched controls. The data highlighted a consistent expression of ApoE deregulated by stress and also by the administration of the CB1 agonist which might indicate a CB1 mediated effect. Therefore, we investigated whether the administration of cannabinoid drugs might have a potential therapeutic effect to overcome the stress-induced cytoskeletal modifications.
Effects of cannabinoid drugs under non-stress conditions
The administration of both drugs reduced ApoE and Rtn4r levels when compared to those subjected to vehicle alone. Here, we demonstrated that alterations in the endocannabinoid signalling by either long-term stress or acute cannabinoid treatment are accompanied by changes in ApoE expression [35, 73]. We reported elevated levels of ApoE in the control group after V+W injection in contrast to those treated with R+W. Similarly, Russell and colleagues (2010) found an increase of ApoE attributable to the drug W [74] while the antagonist acted oppositely [75]. Rtn4r encodes a glycosylphosphatidylinositol-anchored protein remarkably present in the PFC [76] that has been involved in oligodendrocyte proliferation [77], cytoskeleton organization [78], and neuronal processes such as neurotransmission, regeneration, sprouting, and plasticity [79]. A decrease in Rtn4r following R+W treatment was measured when compared to vehicle alone which might act as a compensatory mechanism against functional disruption of the neural PFC system. Indeed, elevated levels of Rtn4r compromise the proper function of PFC [80]. Acute administration of the cannabinoid agonist in non-stressed mice led to an overexpression of Cnp and Mbp when compared to those treated with R+W. CNP participates in RNA splicing, trafficking, and metabolism in mature oligodendrocytes [81–84]. In contrast, MBP regulates the adhesion of compact multilayered myelin sheath [85]. An increase in both myelin-related genes might indicate deficits in myelin CNS architecture [80]. Levels of Vgf, encoding a neuropeptide involved in energy balance, were higher following V+W treatment [86], while the inverse agonist had the opposite effect [87]. This is in line with the results reported upon V+W administration in comparison to R+W. Interestingly, elevated levels of this neuropeptide exacerbates the inflammatory response in rodents [88]. Retinoic acid receptor RXR-alpha (Rxra) participates in the regulation of calcium signalling [89] and synapse formation [90]. Upon activation, RXRA stimulates oligodendrocyte differentiation [91] and promotes the phagocytic functions of microglia [89]. The data presented herein show elevated levels of Rxra following treatment with the CB1 agonist in contrast to R+W. We speculate that this might indicate dysfunctions in oligodendrocyte differentiation, synapse formation, and immune response. The expression of CB1 receptor, encoded by the Cnr1 gene, was higher following V+W than when both drugs were administered in agreement with [80].
Socially defeat mice treated with V+W exhibited lower expression of Mag in comparison to those subjected to vehicle alone. The overall differences observed between these groups could be attributable to the dosage of the cannabinoid agonist applied. An in vitro model of oligodendrocyte differentiation revealed that a low dosage of the cannabinoid agonist confers neuroprotection while the administration of higher doses of the drug aggravates demyelination [92]. Loss of Mag, encoding a transmembrane glycoprotein localized at peri-axonal regions of oligodendroglia, is associated with oligodendrocyte dysfunctions [93]. Under stress conditions, intraperitoneal injection of the cannabinoid antagonist induced a reduction in ApoE levels in comparison to V+W. In contrast, stressed mice subjected to the cannabinoid agonist displayed higher expression of ApoE than their non-stressed counterparts which is in keeping with [74]. Socially defeat mice treated with both cannabinoid drugs underwent elevated levels of ApoE, Cnp, Vgf, Drd1, Drd5, Rxra, Zfp488, Mbp, Cnr1, and Ryr3 in comparison to their non-stressed counterparts (Table 2). Mice subjected to chronic stress and acutely treated with both cannabinoid drugs showed elevated levels of ApoE [73–75]. Co-administration of both drugs under stress resulted in higher levels of Vgf which, in turn, could be attributable to the cannabinoid agonist W [86]. Indeed, exposure to either psychosocial stress or acute administration of the inverse agonist acted in the opposite direction than the cannabinoid agonist did [87, 94]. Dopaminergic neurotransmission is essential for cerebral function, controlling various physiological mechanisms including cognition, locomotion, neuroendocrine activity, emotional and motivational aspects [95]. Recent studies have demonstrated that midbrain dopaminergic neurons are particularly vulnerable to microtubule disruptions [96]. We measured a prominent expression of Drd5 in stressed mice subjected to R+W which could be explained by a stress-mediated effect [97] rather than the consumption of cannabinoid drugs [98]. The administration of R+W under the influence of stress resulted in more Rxra expression than in their non-stressed counterparts. Elevated levels of Rxra might be associated with the use of cannabinoid drugs [35, 99]. There are no data available as yet on the role of Rxra in the context of chronic stress. Subsequently, the function of Rxra under repeated psychosocial stress warrants further investigation. The proliferation of oligodendrocyte precursor cells into myelinating oligodendrocytes is regulated by distinct transcription factors like ZFP488 [100]. We reported an increase of Zfp488 when socially defeat mice were subjected to both cannabinoids in comparison to their non-stressed counterparts [101]. This fact might indicate dysfunctions in oligodendrocyte differentiation [100]. Upon coadministration with R+W, stressed mice displayed higher Mbp expression than their control counterparts indicating perturbations in CNS myelination [85]. These alterations could be explained by the administration of synthetic cannabinoid drugs [80] rather than exposure to a repeated stressor [102]. The expression of Cnr1 was higher in stressed mice subjected to R+W than their controls subjected to equal pharmacological treatment. Such increase could be attributed to either drug treatment [103] or exposure to chronic stress [104]. Ca2+ release into the cytoplasm of neurons is regulated by ryanodine receptors [105]. Changes in RYR3 activity lead to an imbalance in intracellular levels of calcium contributing to an impairment in neurotransmission [106] and lastly neurodegeneration [107]. We reported higher expression of Ryr3 in social defeat mice treated with R+W than their matched non-stressed mice. This evidence might be explained by either chronic exposure to a stressor [108] or acute cannabinoid administration [109].
Conclusions
In summary, we concluded that the decrease in acetylated MTs most likely affects neurotransmission, which nevertheless could promote neuroprotection under long-term stress conditions. Additionally, enhanced HDAC6 activity attributable to lower levels of tubulin acetylation could promote autophagy and clearance of misfolded proteins and aggregates in stressed animals [6]. In this regard, we can speculate that an increase of ApoE under the influence of stress might contribute to neuroprotection as well. It has been shown that ApoE participates in MT polymerization and neurite extension [110, 111]. HDAC6 affects both the nuclear localization of ApoE and the microtubule-organizing center [112]. Thus, elevated levels of ApoE together with a decrease in tubulin acetylation might indicate a dynamic reassembling of the MTs. Collectively, the overexpression of ApoE, CxCl12-ɣ, Dtnbp1, and Cnr1 in the PFC of stressed animals may trigger MT destabilization while tubulin deacetylation could promote MT reorganization acting as a protective mechanism against the side effects of stress. We found a consistent expression of ApoE attributable to either psychosocial stress [65, 66] or administration of the CB1 agonist [74]. Interestingly, the expression of ApoE was inversely correlated with acetylated tubulin levels when comparing controls and stressed mice subjected to the CB1 agonist whereas the use of the CB1 inverse agonist acted oppositely. This fact might indicate a CB1 receptor-mediated effect. The diverse effects on tubulin acetylation observed in the studied brain regions of both control and stressed animals with or without pharmacological treatment might indicate that HDAC6 activity is differently regulated in the brain acting simultaneously on different cellular mechanisms to safeguard homeostatic processes for the proper function of the CNS.