The dynamic rearrangement of the neuronal MTs is crucial for brain plasticity by enabling the remodelling of dendrites and axons 43. 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 and the DS, whereas non-significant differences were observed in the HIPP 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.
Neuronal remodelling and brain plasticity are regulated by the MT system. In animals, stressful conditions can impair neuronal plasticity. Stress-induced neuronal atrophy in the HIPP by retraction of apical dendrites, cell death, and decreased neurogenesis 4,47−49. However, it is not restricted to this brain region. In particular, chronic restraint stress in experimental animals also provoked atrophy in the PFC 46,50. Since acetylation of tubulin contributes to cytoskeletal stability, a decrease might lead to synaptic and dendrite alterations 48. Consistently, we found a decreased tubulin acetylation in the PFC and the DS derived from mice exposed to psychosocial stress.
Our results indicate an imbalance of tubulin acetylation and deacetylation in social defeat mice pointing to either reduced expression or inhibition of the enzymatic activity of the acetyltransferase Atat1 or hyper-activity of the HDAC6 tubulin deacetylase. A change in gene expression, neither for Atat1 nor Hdac6, was not assessed by Nanostring gene expression arrays. However, it is well known that ATAT1 is highly expressed in the cerebral cortex and the HIPP (retrieved from The Human Protein Atlas at https://www.proteinatlas.org/ENSG00000137343-ATAT1/tissue), whereas HDAC6 is highly expressed in the brain and especially in the cortex, the HIPP, and the CRB (49; retrieved from The Human Protein Atlas at https://www.proteinatlas.org/ENSG00000094631-HDAC6/tissue). Since HDAC6 is the main α-tubulin deacetylase in the brain, changes in tubulin acetylation are most likely due to the deregulation of HDAC6 activity. HDAC6, additionally, targets also the chaperone Hsp90 and the redox regulatory proteins peroxiredoxin I and II (Prx I and II), both of them involved in the stress response 21,50,51. Perturbation of acetylated tubulin dynamics has been reported in neuropsychiatric diseases and stress-related disorders 8–10. Furthermore, HDAC6 affects the emotional behaviour of experimental animals since Hdac6-deficient mice exhibit hyperactivity, less anxiety, and anti-depressant-like behaviour 27. Cells respond to stress immediately with the formation of cytoplasmic stress granules by recruitment of HDAC6. Interestingly, an appropriate activity of HDAC6 is required to counteract oxidative stress 52. Besides that, HDAC6 is essential for the clearance of misfolded proteins and protein aggregates that are degraded by either the ubiquitin-proteasome pathway or autophagy. Indeed, HDAC6 binds to polyubiquitinated misfolded proteins favouring their retrograde MT transport to aggresomes. Thus, HDAC6 is a component of aggresomes that increases the efficiency and selectivity of autophagic degradation and protects cells from stress response caused by aggregation of misfolded proteins 22,53. Subsequently, an increase of HDAC6 activity could promote autophagy and clearance of misfolded proteins and protein aggregates, e.g., aggregates of hyperphosphorylated Tau proteins generated by chronic stress 6. A decrease in tubulin acetylation as observed in the PFC and the DS of the stress group could thus indicate a neuroprotective function attributable to the overactivation of HDAC6. Elevated levels of HDAC6 deacetylase activity are under the tight control of the kinases Aurora A and GSK3-ß 54,55. Activation of Aurora A is regulated by HEF1 (human enhancer of filamentation 1) that in turn is activated by HIF-1α (hypoxia-inducible factor 1α) 56. Thus, putatively, stressful events might induce hypoxic conditions that favour Aurora A and HDAC6 activation and lastly, tubulin deacetylation. Blockage of GSK3-ß inhibits deacetylase activity and simultaneously increases tubulin acetylation. GSK3-ß is a substrate of the PI3K/AKT pathway and becomes phosphorylated and inactivated by stimulation of AKT. Thus, the inhibition of the AKT pathway activates HDAC6 reducing tubulin acetylation. Additionally, GSK3-ß negatively regulates mitochondrial and anterograde axonal transport 55,57. The activity of the PI3K/AKT pathway is controlled by upstream molecules such as the CXCL12-CXCR4 and DTNBP1. Drug-induced tubulin acetylation and MT stabilization are suppressed upon CXCL12 activation in prostate cancer cells 58. Dysbindin-1 regulates synapse morphology, synaptic plasticity, vesicle trafficking, and reduced dopamine-induced phosphorylation of AKT/GSK3ß 59. Thus, elevated levels of CxCl12-ɣ or Dtnbp1, as observed in the PFC of stressed animals, might contribute to HDAC6 hyperactivation and eventually tubulin deacetylation.
In the HIPP, the DS, and the CRB, activation of the G-protein coupled cannabinoid receptor CB1, by administration of Δ9-tetrahydrocannabinol (THC), increased phosphorylation of AKT, whereas the selective CB1 antagonist R blocked it 60. Furthermore, the phosphorylation of GSK3-ß was also increased by THC administration 60. We showed here, that the administration of the CB1 agonist increased tubulin acetylation in the PFC of stressed animals. In contrast, a reduction or no significant changes were found in the HIPP, the DS, and the CRB of the control and the stress group when the same drug was administered. As expected, activation of CB1 by use of the cannabinoid agonist activates AKT kinase that in turn inactivates GSK3-ß by phosphorylation eventually blocking the activity of HDAC6 resulting in elevated levels of tubulin acetylation. Such effect was observed exclusively in the PFC. We reported higher expression of Cnr1 in the PFC of stressed animals that could prevent the hyperactivation of HDAC6 via PI3K/AKT signaling. Instead, we observed a decrease in tubulin acetylation levels pointing out that the overexpression of Cnr1 measured here might act as a compensatory mechanism to counteract the reduced levels of tubulin acetylation. However, further analyses warrant investigation. Collectively, our results demonstrate that distinct brain regions respond differentially to either prolonged stress or the administration of cannabinoid drugs. Furthermore, the different levels of tubulin acetylation reported here could suggest that neuroprotection mediated by CB1 is quite variable and cell type-specific. In summary, our findings revealed a reduction of tubulin acetylation under chronic stress when the PFC and the DS were studied. This fact could be an indicator of HDAC6 hyperactivity. Dysfunctions in tubulin acetylation under stress conditions might indicate that cellular architecture and vesicle transport are compromised 14,15. But taking into account the protective role of HDAC6 against cellular stress, an alternative scenario could suggest that changes in tubulin acetylation could confer protection against chronic stress instead of being just a detrimental outcome.
Chronic psychosocial stress, such as the social defeat model, caused a variety of molecular, physiological, and behavioral changes 34,35,39. Upon repeated psychosocial stress, differential gene expression was found in the CRB and other brain regions 35,61. Elevated levels of ApoE, CxCl12ɣ, Dtnbp1, Ski, and Cnr were found in stressed mice subjected to V+V when compared to their matched controls (Table 2). As reported, most of these genes are related to neuronal functioning. ApoE is involved in lipid metabolism and acts as the principal cholesterol carrier in the brain 62. Experimental and clinical studies both revealed that ApoE levels are related to stress response 63,64. Furthermore, repeated psychosocial stress per se stimulates inflammatory response by activating leukocyte extravasation through the blood-brain barrier into the brain 65. This process is tightly regulated by the complex Cxcl12-Cxcr4. Repeated social defeat promotes the synthesis of CxCl12 within the brain exacerbating the pro-inflammatory reaction 66 in line with the results reported here. Dtnbp1 is involved in distinct biological processes, including dendritic spines and synapse formation 67. Overexpression of Dtnbp1 under stress conditions might confer vulnerability to psychotropic drugs 68. Ski is a transcriptional modulator required for the expansion of precursor cells in the neuroepithelium or skeletal muscle lineages 69. Ski has been involved in distinct biological processes such as muscle homeostasis, axonal growth, myelination, hematopoietic cell differentiation, regulation of T-cells as well as in many complex pathologies 70. However, its function in the context of prolonged stress remains to be elucidated and warrants further investigation.
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 35,71. 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 72 while the antagonist acted oppositely 73. Rtn4r encodes a glycosylphosphatidylinositol-anchored protein remarkably present in the PFC 74 that has been involved in oligodendrocyte proliferation 75, cytoskeleton organization 76, and neuronal processes such as neurotransmission, regeneration, sprouting, and plasticity 77. 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 78. 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 79–82. In contrast, MBP regulates the adhesion of compact multilayered myelin sheath 83. An increase in both myelin-related genes might indicate deficits in myelin CNS architecture 78. Levels of Vgf, encoding a neuropeptide involved in energy balance, were higher following V+W treatment 84, while the inverse agonist had the opposite effect 85. This is in line with the results reported upon V+W administration in comparison to R+W. Interestingly, elevated levels of this neuropeptide exacerbated the inflammatory response in rodents 86. Retinoic acid receptor RXR-alpha (Rxra) participates in the regulation of calcium signalling 87 and synapse formation 88. Upon activation, RXRA stimulates oligodendrocyte differentiation 89 and promotes the phagocytic functions of microglia 87. 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 78.
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 90. Loss of Mag, encoding a transmembrane glycoprotein localized at peri-axonal regions of oligodendroglia, is associated with oligodendrocyte dysfunctions 91. 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 72. 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 71–73. Co-administration of both drugs under stress resulted in higher levels of Vgf which, in turn, could be attributable to the cannabinoid agonist W 84. Indeed, exposure to either psychosocial stress or acute administration of the inverse agonist acted in the opposite direction than the cannabinoid agonist did 85,92. Dopaminergic neurotransmission is essential for cerebral function, controlling various physiological mechanisms including cognition, locomotion, neuroendocrine activity, emotional and motivational aspects 93. Recent studies have demonstrated that midbrain dopaminergic neurons are particularly vulnerable to microtubule disruptions 94. We measured a prominent expression of Drd5 in stressed mice subjected to R+W which could be explained by a stress-mediated effect 95 rather than the consumption of cannabinoid drugs 96. 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 35,97. 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 98. We reported an increase of Zfp488 when socially defeat mice were subjected to both cannabinoids in comparison to their non-stressed counterparts 99. This fact might indicate dysfunctions in oligodendrocyte differentiation 98. Upon coadministration with R+W, stressed mice displayed higher Mbp expression than their control counterparts indicating perturbations in CNS myelination 83. These alterations could be explained by the administration of synthetic cannabinoid drugs 100 rather than exposure to a repeated stressor 101. 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 102 or exposure to chronic stress 103. Ca2+ release into the cytoplasm of neurons is regulated by ryanodine receptors 104. Changes in RYR3 activity lead to an imbalance in intracellular levels of calcium contributing to an impairment in neurotransmission 105 and lastly neurodegeneration 106. 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 107 or acute cannabinoid administration 108.
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 109,110. HDAC6 affects both the nuclear localization of ApoE and the microtubule-organizing center 111. 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 63,64 or administration of the CB1 agonist 72. 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.