Study of autophagic and epigenetic mechanisms in experimental models of inflammatory and neuropathic pain for the identification of new pharmacological targets

Abstract

Pain is defined by IASP as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP, 2011). While physiological pain is like a warning system, useful to prevent damage to the organism, pathological pain is an unpleasant sensation, permanent also after damage and it is characterized by an enhanced sensitivity to both innocuous and noxious stimuli (termed allodynia and hyperalgesia, respectively). While acute pain resolves in few days, chronic pain lasts longer than three/six months. Neuropathic pain, a common form of chronic pain, was defined as “pain caused by a lesion or disease of the somatosensory nervous system” (IASP, 2011). Pharmacological treatments available, including tricycles antidepressant and gabapentin, have limited efficacy in most of patients (Childers et al, 2007). Therefore, a better understanding of pain physiopathology and the development of new treatments are very important. Here, we characterised two new molecular mechanisms, autophagy and epigenetic mechanisms, and their role in pain processing. Autophagy is the main mechanisms involved in the degradation of proteins and organelles, in cell remodelling and survival during periods of nutrient deficiency. The decrease in the autophagic activity seems to interfere with the degradation of proteins and with the turnover of nutrients, while a greater activation of this pathway appears to facilitate the clearance of protein aggregates and to promote neuronal survival in various neurodegenerative diseases. On the other hand, too high autophagic activity can be detrimental and lead to cell death, suggesting that the regulation of autophagy has an important role in determining cell fate. However, despite numerous studies on the role of autophagy in neurodegenerative diseases, the role of this process in the pathophysiology of neuropathic pain remains poorly studied. Epigenetic mechanisms are chemical modifications of chromatin that influence gene expression without altering the DNA sequence. Although in recent years scientific research has produced significant results in the epigenetics field, only few studies have focused on the involvement of epigenetic mechanisms in relation to pain states. Experimental evidence suggests that changes in the expression of some genes are involved in the early stages of induction and maintenance of chronic pain states. Among these genes, recent evidence suggests a role for the FKBP5 gene, an important regulator of the glucocorticoid receptor, involved in the regulation system of the stress response. In addition, recent studies show that this gene is under strong epigenetic control. In view of this, the objectives of this research were: • To characterise the autophagic process at spinal cord level in different experimental models of neuropathic and inflammatory pain; • To verify the relevance of spinal autophagy for pain processing; • To identify pain conditions in which the gene FKBP5 plays a role; • To study the role of FKBP5 on pain processing at spinal cord level; • To characterize the enzymes involved in DNA methylation; The results obtained in the first experimental part of this thesis showed a modulation of the main autophagic markers in experimental models of neuropathic pain. In particular, in the model that involves the ligation of the L5 spinal nerve (SNL) and in the model that involves the transection of the tibial nerve and peroneal (SNI), it was observed an increase in the levels of the associated form of the protein LC3 (LC3II ) and of protein p62 , which is involved in the early stages of degradation of the autophagic process. The observed increase in p62 protein levels suggested a possible impairment of autophagic flux. To verify this hypothesis the consequences of a local block of autophagy at spinal level were investigated on pain behaviour. In particular, the treatment of naïve animals with chloroquine, a lisosomal inhibitor, resulted in the establishment of a state of hyperalgesia typically observed after peripheral damage of the spinal nerves. The results obtained in the second experimental part demonstrate an involvement of the gene FKBP5 in the induction and in the maintenance phases of chronic pain. In particular, knockout animals have shown a lower sensitivity to mechanical stimuli following the onset of various chronic pain states. The silencing of the gene at the spinal cord level has allowed us to understand the role of the gene FKBP5 in pain processing after an injury. Finally, the study and characterization of DNMT1, the enzyme involved in DNA methylation, has allowed us to suggest the active involvement of other proteins in the process of DNA demethylation and then in the expression of genes. In conclusion, the data reported in this study indicate an impairment of autophagy in experimental models of neuropathic pain, supporting the neuroprotective role of this process in the spinal cord. It was also demonstrated the involvement of the gene FKBP5 in the induction and in the maintenance phases of chronic pain. Altogether, these data pave the way to further investigations aimed to a better understanding of the mechanisms underling chronic pain and to the identification of potential molecular targets for the development of new therapeutic strategies