RESUMO
Although peripheral neuropathic pain is caused by peripheral nerve injury, it is not simply a peripheral nervous system disease. It causes abnormalities in both the central and peripheral nervous systems. Pathological phenomena, such as hyperactivation of sensory neurons and inflammation, are observed in both the dorsal root ganglion and spinal cord. Pain signals originating from the periphery are transmitted to the brain via the SC, and the signals are modulated by pathologically changing SC conditions. Therefore, the modulation of SC pathology is important for peripheral NP treatment. We investigated the effects of KLS-2031 (recombinant adeno-associated viruses expressing glutamate decarboxylase 65, glial cell-derived neurotrophic factor, and interleukin-10) delivered to the dorsal root ganglion on aberrant neuronal excitability and neuroinflammation in the SC of rats with peripheral NP. Results showed that KLS-2031 administration restored excessive excitatory transmission and inhibitory signals in substantia gelatinosa neurons. Moreover, KLS-2031 restored the in vivo hypersensitivity of wide dynamic range neurons and mitigated neuroinflammation in the SC by regulating microglia and astrocytes. Collectively, these findings demonstrated that KLS-2031 efficiently suppressed pathological pain signals and inflammation in the SC of peripheral NP model, and is a potential novel therapeutic approach for NP in clinical settings. PERSPECTIVE: Our study demonstrated that KLS-2031, a combination gene therapy delivered by transforaminal epidural injection, not only mitigates neuroinflammation but also improves SC neurophysiological function, including excitatory-inhibitory balance. These findings support the potential of KLS-2031 as a novel modality that targets multiple aspects of the complex pathophysiology of neuropathic pain.
Assuntos
Neuralgia , Doenças Neuroinflamatórias , Ratos , Animais , Neuralgia/terapia , Medula Espinal , Terapia Genética , Inflamação , Células Receptoras Sensoriais , Hiperalgesia , Gânglios EspinaisRESUMO
Neuropathic pain is a chronic pain state characterized by nerve damage, inflammation, and nociceptive neuron hyperactivity. As the underlying pathophysiology is complex, a more effective therapy for neuropathic pain would be one that targets multiple elements. Here, we generated recombinant adeno-associated viruses (AAVs) encoding three therapeutic genes, namely, glutamate decarboxylase 65, glial cell-derived neurotrophic factor, and interleukin-10, with various combinations. The efficacy for pain relief was evaluated in a rat spared nerve injury model of neuropathic pain. The maximal analgesic effect was achieved when the AAVs expressing all three genes were administered to rats with neuropathic pain. The combination of two virus constructs expressing the three genes was named KLS-2031 and evaluated as a potential novel therapeutic for neuropathic pain. Single transforaminal epidural injections of KLS-2031 into the intervertebral foramen to target the appropriate dorsal root ganglion produced notable long-term analgesic effects in female and male rats. Furthermore, KLS-2031 mitigated the neuroinflammation, neuronal cell death, and dorsal root ganglion hyperexcitability induced by the spared nerve injury. These results suggest that KLS-2031 represents a promising therapeutic option for refractory neuropathic pain.
RESUMO
Deubiquitination via deubiquitinating enzymes (DUBs) has been emerged as one of the important post-translational modifications, resulting in the regulation of numerous target proteins. In this study, we screened new protein biomarkers for adipogenesis, and related studies showed that ubiquitin specific protease 19 (USP19) as a DUB is gradually decreased during adipogenesis and it regulates coronin 2A (CORO2A) as one of the components for the nuclear receptor co-repressor (NCoR) complex in some studies. The regulation of CORO2A through the deubiquitinating activity of USP19 affected the transcriptional repression activity of the retinoic acid receptor (RAR), suggesting that USP19 may be involved in the regulation of RAR-mediated adipogenesis.
Assuntos
Adipogenia/genética , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Proteínas dos Microfilamentos/genética , Processamento de Proteína Pós-Traducional/genética , Receptores do Ácido Retinoico/genética , Adipogenia/fisiologia , Animais , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/antagonistas & inibidores , Transcrição Gênica/genéticaRESUMO
HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage.
Assuntos
Dano ao DNA/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética , Anexina A1/genética , Anexina A1/metabolismo , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina , NucleolinaRESUMO
Ubiquitination and deubiquitination are important processes for numerous intracellular mechanisms, and the imbalance of these two processes can cause severe diseases including cancer. Accordingly, deubiquitinating enzymes (DUBs) responsible for deubiquitination from their protein substrates become attractive targets for many studies. USP20, also known as VDU2, belongs to ubiquitin-specific protease (USP) subfamily of DUBs and has several important roles in cells as shown with other DUBs. USP20 stabilizes HIF-1α by abolishing von Hippel-Lindau protein (pVHL)-E3 ligase complex-mediated HIF-1α degradation. USP20 is also associated with ß2 adrenergic receptor recycling. In addition, a previous study demonstrated that USP20 regulates Tax-induced NF-κB activation through its deubiquitinating activity. These studies provide a line of evidence that USP20 has critical roles in cellular functions. In this study, we generated and characterized a polyclonal and two monoclonal antibodies against USP20. It is feasible that USP20 antibodies can be useful to investigate USP20-related cellular mechanisms and to find novel substrates of USP20.