| dc.description.abstracteng | Chronic pain imposes substantial challenges to medical practice as the treatment options for its clinically relevant manifestations are limited. Owing to lack of knowledge about the exact molecular mechanism underlying pathological pain conditions, pain therapeutics currently available target molecules with key physiological functions in our body. Thus, they are accompanied by severe side effects, which limits effective dosage prescription. Bearing these difficulties in mind, it is highly desirable to identify the proteins and their associated complexes that are differentially regulated and function at the forefront of noxious stimulus detection. The goal of this study was to identify and characterize multiprotein complexes in the context of nociception in mice. On the one hand, I followed a unbiased mass spectrometry (MS)-based approach to characterize the changes in expression of a large set of proteins in the dorsal root ganglia (DRG). DRG harbour the somata of nociceptors, the primary afferent sensory neurons that express distinct molecular sensor of painful stimuli. On the other hand, I aimed to reveal the scaffold of proteins interacting with the Transient Receptor Potential V1 (TRPV1) ion channel, a polymodal sensor of irritant chemicals and noxious heat.
In order to elucidate the molecular underpinnings of chronic pain, several large-scale profiling studies have been performed. However, the generated lists of regulated candidate proteins are often inconsistent with scarce overlap. This could be explained by inherent technical limitations of used proteomics methods, such as low reproducibility. Emerging data-independent acquisition mass spectrometry (DIA-MS) has the potential to allow for standardized and reproducible quantification across many samples. Here, we applied DIA-MS on DRG isolated from mice subjected to two mouse models of chronic pain to define global changes in the DRG proteome. More specifically, we compared the abundance of 2,526 DRG proteins across the two pain models and their respective controls. Considerable and pain-model specific alterations in the abundance of several dozen proteins as well as within functional protein networks were detected. These were validated with several orthogonal methods. Amongst others, the analysis of mouse pain behaviours verified that meaningful protein alterations both on the level of single proteins and signalling networks were revealed with our workflow.
The involvement of TRPV1 in different chronic pain states has been well documented and together with its enriched expression in DRG renders TRPV1 a promising target for novel analgesics. However, several TRPV1 antagonists that reached clinical trials are challenged by severe side effects because of interference with physiological functions of TRPV1. An interesting alternative to TRPV1 blockage might be the targeting of such TRPV1 interaction partners that are specific for e.g. inflammatory pain. This strategy would provide a means to suppress pathological pain states whilst leaving nociceptive pain intact. However, very little is known about the protein scaffold of TRPV1 during different pain states. Here, I present the Vesicle transport through interaction with t-SNAREs homolog 1B (Vti1b) as a novel pain-specific interactor of TRPV1. Vti1b modulates TRPV1 sensitization within an inflammatory milieu in vitro. Normal functioning of the TRPV1 is left intact. In vivo virus-mediated knockdown of Vti1b diminished the development of thermal hypersensitivity upon CFA injection in mice. The knockdown does not affect CFA-evoked mechanical hypersensitivity or capsaicin-induced nocifensive behaviour. In a second step, a functional proteomics approach was employed to identify the TRPV1 interactome under CFA-induced inflammatory pain in mouse DRG neurons. Comparison of the interactomics data between the control and CFA group revealed a significant regulation of the TRPV1 interactome upon induction of inflammatory pain. For instance, Vti1b was found to be less abundant in TRPV1 protein complexes upon inflammation. Overall, this study strongly supports the notion that protein-protein interactions specific for pathological pain exist.
In summary, these two mass spectrometric studies represent a unique resource on (I) the differential expression of membrane proteins during pathological pain and (II) the dynamics of TRPV1 interactors during inflammatory pain. Acquired data may contribute to the characterization of the molecular mechanisms underlying pathological pain and may therefore facilitate the development of more effective therapeutic strategies. | de |