The role of Proteasome in the development, maintenance and management of Chronic Pain

In chronic pain, opioids represent the gold standard analgesics, but their use is hampered by the development of several side effects, as development of analgesic tolerance and opioid-induced hyperalgesia. Evidence showed that many molecular mechanisms (changes in opioid receptors, neurotransmitter release, and glia/microglia activation) are involved in their appearance, as well as in chronic pain. Recently, a crucial role has been proposed for oxidative stress and proteasome in chronic pain and in treatment-related side effects. To better elucidate these aspects, the aim of this PhD thesis was to investigate the effects of opioids on cell oxidative stress, antioxidant enzymatic machinery and proteasome expression and activity in vitro. Also, the involvement of proteasome in the development of chronic pain conditions was investigated utilizing an experimental model of oxaliplatin-induced neuropathy (OXAIN), in vivo. Data showed that morphine, fentanyl, buprenorphine and tapentadol alter differently ROS production. The ROS increasing effect of morphine is not shared by the other opioids, suggesting that the different pharmacological profile could influence this parameter. Moreover, these drugs produced different alterations of β2trypsin-like and β5chymotrypsin-like activities. In fact, while morphine and fentanyl increased the proteolytic activity after prolonged exposure, a different picture was observed for buprenorphine and tapentadol, suggesting that the level of MOR agonism could be strongly related with proteasome activation. In vivo studies revealed that rats treated with oxaliplatin showed a significant increase in β5, in the thalamus (TH) and somatosensory cortex (SSCx). Moreover, a selective up-regulation of β5 and LMP7 subunit gene expression was assessed in the SSCx. Furthermore, our study revealed that oprozomib, a selective β5 inhibitor normalized the spinal prodynorphin gene expression upregulation induced by oxaliplatin, and reverted mechanical/thermal allodynia and mechanical hyperalgesia in oxaliplatin-treated rats. These results underline the role of proteasome in the OXAIN and suggest new pharmacological targets to counteract it.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.