Optimizing neuron-specific knock-down techniques to investigate the role of Importin-α3 in pain and neuronal development in Caenorhabditis elegans

Importin-α proteins, members of the Karyopherin super family, play a crucial role in regulating nucleus-cytoplasmic transport by recognizing nuclear localization signals (NLS) on cargo proteins. This role is particularly significant in the context of development where Importin-αs are acting as gatekeepers of the genome. They selectively facilitate the entry of transcription factors into the nucleus which in turn are activating gene expression programs that drive cellular differentiation, such as neuronal specialization. Neurons are terminally differentiated cells with highly specialized functions that rely on the correct morphogenesis and axonal pathfinding for proper neural circuits formation. While seven Importin-α proteins are expressed in humans and their molecular mechanisms are well characterized, their functional roles and specific binding partners remain largely unexplored. There is growing interest in their potential organismal functions. For instance, Importin-α3 was recently shown to have a functional role in pain signaling. We therefore focused on Importin-α3, aiming to establish all the tools necessary to study Importin-α3 using a systems approach and verify whether its role and functions in pain signaling are evolutionary conserved across species. Given the complexity of the human and vertebrate models nervous system, dissecting the functions of Importin-α in specific neuronal populations is quite challenging. In contrast, the nematode Caenorhabditis elegans offers a powerful alternative due to its well-mapped nervous system, short life-cycle, and suitability for large-scale genetic studies. Its unique neuron genetics, allows for genetically targeting single neurons, which provides unparalleled insights into neuronal differentiation and specialization. We, therefore, aim to generate neuron-specific knock-down strains lacking Importin-α3 expression in specific sensory neurons: ASH, FLP and PDV. To achieve this, we refined a C.elegans-specific RNAi knock-down technique. To obtain our sequence of interest, we carefully optimized a fusion-PCR-based cloning approach to link the ima-3 sense and antisense sequences, with a neuron-specific promoter. When injected into the gonad of the animal the presence of ima-3 sense and antisense sequences creates neuron-specific knock-down of the endogenous ima-3. Similarly, we attempted to link the GFP sequence to an ASH-neuron-specific promoter. To test the effects of ima-3 knock-down in ASH neurons, we developed a pH-gradient-based assay that relies on fully functional ASH neurons. We carefully optimized the pH-gradient, osmolarity and temperature homogeneity to ensure maximum sensitivity toward even mild ASH deficiencies. Finally, we demonstrated the effectiveness of this assay on ASH-ablation strain. Together this assay and the knock-down strains will be used to determine the effects of ima-3 loss on pain sensation and behavior. In future, confocal laser scanning microscopy will be used to measure neuronal morphology across developmental stages, allowing us to examine Importin-α3’s potential role in neuronal differentiation, axon guidance and synapse formation. By comparing neuronal structure and function, we aim to map the role of importins in neuron development and mature neurons defining their role in health and diseased states. Our efforts will determine whether importins have evolutionary-conserved, specific organismal functions such as regulation of pain-specific gene expression. Gaining function-specific access to the neural genome will enable more specific genetic interventions in the nervous system.