Characterization of novel protein players in pain
by Hanna Kristina Fischer
Date of Examination:2023-02-23
Date of issue:2023-08-16
Advisor:Dr. Manuela Schmidt
Referee:Dr. Manuela Schmidt
Referee:Prof. Dr. Thomas Dresbach
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EnglishDespite its high prevalence in the human society, the mechanisms underlying chronic pain and pain chronification are still poorly understood. The shortage of potential targets that are specific to pathological chronic pain, as opposed to the physiologically necessary acute pain, lead to limited treatment options. With the few treatment options that are available, it often comes to a lack of efficacy and a high rate of side effects, including addiction, especially in the case of opioids. Additionally, it is well described that pain perception and the underlying mechanisms differ immensely depending on age and sex of the subject, which is usually not considered for treatment. A possible method to improve treatment options in the future is targeting molecules relevant to chronic pain and specific to the different phenotypes and characteristics of the subjects. Therefore, it is of high importance to understand mechanistical differences between acute pain and different types of chronic pain in more detail, but also between sexes and juvenile and adult individuals. Different experimental concepts were applied throughout this thesis to enhance the understanding of the processes underlying pain. In a first section, one specific protein, Tmem160, was targeted by generating a knockout (KO) mouse line to investigate its role in chronic pain. This study also focused on the influence of sex within this context. Tmem160 was chosen as a promising target, as this hitherto undescribed protein was recently shown to be downregulated in murine chronic pain models. The Tmem160 KO mice were subjected to multiple pain models and compared to their wildtype (WT) littermates. Under neuropathic pain condition, as caused by the spared nerve injury (SNI) model, deletion of Tmem160 elicited a concise phenotype in male mice in the initiation phase, namely a delay in the development of tactile hypersensitivity and an absence of increased self-grooming behavior in contrast to WT littermates. Also, the function of Tmem160 displayed a sexual dimorphism: female Tmem160 KO mice presented WT-like behavior within the SNI model. Furthermore, mouse models for acute, incisional and inflammatory pain showed that Tmem160 is dispensable under these conditions, indicating an important specificity to neuropathic pain. Possible action mechanisms of Tmem160 were investigated using in vitro experiments. Male Tmem160 KO mice revealed an overall reduction of cytokine levels in naïve DRG that potentially influenced neuronal excitability by reducing the percentage of cells responding to activation of the Transient receptor potential ankyrin 1 (TRPA1) channel. This reduction compared to WT could be restored by previous incubation with Tumor necrosis factor-α (TNFα), one of the cytokines found to be downregulated. Investigation of a marker for microglia/macrophages in spinal cord, indicates a reduction in spinal activation of these cells upon nerve injury. Additionally, a conditional knockout (cKO) of Tmem160 in primary sensory neurons suggests non-neuronal cells as the primary site of action for Tmem160 in the chronic pain context, since the cKO showed WT-like behavior and characteristics, implying that the neuronal Tmem160 is dispensable for the observed processes. Taking together, a hypothetic working model for its action mechanism was developed, with non-neuronal Tmem160 influencing neuroimmune crosstalk in the DRG in the early phase of neuropathic pain: It interacts with TRPA1 expression levels, conceivably via TNFα and affects neuronal excitability of the primary sensory neurons. The non-neuronal Tmem160 is most likely relevant in microglia/macrophages, also explaining the sex-differences related to the higher relevance of these cells for pain pathways in males compared to females. The investigation of specific individual characteristics was even more important in the second study where the influence of age and sex on somatosensation and on the proteomic composition of the dorsal root ganglia (DRG), a tissue crucial for somatosensation, was investigated. Under naïve conditions, male juvenile mice showed a decrease in thermal sensitivity throughout maturation, while the nocifensive response within a model for acute pain increased with age. To investigate underlying mechanisms in vitro experiments were performed on DRG. Transient receptor potential vanilloid 1 (TRPV1) channel expression levels, as well as the neuronal excitability in response to TRPV1 stimulation, revealed differences depending on age. The proteome of the DRG under naïve conditions was studied and compared between juvenile and adult, male and female mice using quantitative proteomics. Many proteins showed an age-dependent differential expression, including multiple candidates involved in pain and neuroimmune interactions. The number of age-dependent differentially expressed proteins (DEPs) was significantly higher in female than in male mice. When comparing the sexes directly within the age groups very few differences in the proteome were detected. This shows that maturation and age have a stronger influence on the composition of the DRG proteome than sex. Even though the composition of the DRG does not differ a lot between sexes within age groups, it must be acknowledged that the changes occurring during maturation showed a difference between sexes. In addition to the identification of Tmem160 as a promising candidate to specifically target chronic pain, while leaving acute pain functions intact, this study underlines the need of an adequate selection of animals in pain research, both regarding their sex and age. It shows the importance to match the characteristics of the research animals with the impacted group of society. A better matching of characteristics has the power to improve translation between animals and humans to enhance and enable targeted treatment options at the level of individuals and their specific disease.
Keywords: chronic pain; neuropathic pain; mouse pain behavior; maturation; somatosensation; cytokines; pain initiation; neuro-immune interaction; nerve injury; dorsal root ganglia; inflammatory signaling; sex differences