GCAP1 in Autosomal Dominant Cone-Rod Dystrophy: A Multifaceted Biochemical Investigation and Therapeutic Perspectives

Vision, the sense that enables light perception and image for mation, begins with the intricate process of phototransduc tion in the retina. This biochemical cascade converts pho tons into electrical signals, triggering sight. Central to this process are pho toreceptor cells, rods and cones, which employ a complex array of proteins to transduce light into neural signals. Among these proteins, guanylate cyclase activating proteins (GCAPs) and retinal degeneration protein 3 (RD3) play a crucial role in modulating the vision process. By sensing subtle changes in intracellular Ca2+ levels, GCAP dimers regulate the activity of guanylate cy clases (GCs), enzymesresponsibleforsynthesizingcyclicguanosinemonophos phate (cGMP). Within human photoreceptors, GCAP1 appears to be the main actively participating in this process, serving as a regulator of GC1 isozyme. RD3 further modulates this pathway by vehiculating GC1 from the inner to the outer segment of photoreceptors and strongly inhibiting its activity, thus rep resenting a critical regulatory factor in the phototransduction cascade. This concerted regulation is essential for maintaining the homeostasis of cGMP and Ca2+ levels, crucial second messengers in photoreceptors, ensuring the proper function of the visual process. Studying GCAP1 and its interactions within the phototransduction pathway is vital for understanding the funda mental mechanisms underlying vision and the pathogenesis of inherited reti nal dystrophies (IRDs) caused by mutations in this protein. Disruptions in the GCAP-mediated feedback mechanism can lead to abnormal photorecep tor responses and, ultimately, progressive vision loss. Therefore, investigat ing the biochemical and biophysical characteristics of GCAP1 variants and their impact on photoreceptor functionality provides critical insights into the biochemical signatures of IRDs. This thesis investigates the molecular ba sis and therapeutic avenues for autosomal dominant cone dystrophy (adCOD) and cone-rod dystrophy (adCORD), focusing on mutations in the GUCA1A gene coding for GCAP1. Through biochemical and computational investi gations it elucidates the impacts of the N104H-GCAP1 and E111V-GCAP1 variants on photoreceptor functionality. The N104H mutant, characterized by a novel missense mutation in GUCA1A, reveals a unique biochemical profile with diminished calcium sensitivity and doubled affinity for retinal guanylate cyclase 1 (GC1), diverging from previously studied mutations and suggest ing a mechanism for photoreceptor cell degeneration through aberrant cGMP and calcium accumulation. On the other hand, the E111V variant maintains the monomer-dimer equilibrium essential for phototransduction, despite its constitutive activation of GC1, indicating the mutation’s specific impact on enzyme stimulation rather than the dimerization processes. Molecular dock ing and dynamics simulation highlight the subtle alterations induced by the E111V substitution, offering insights into its enhanced mobility and altered interaction with GC1. Moreover, the strong inhibitory activity of RD3 was successfully exploited to modulate the abnormal cGMP production induced by the E111V variant, demonstrating RD3’s potential in restoring photoreceptor cell homeostasis. In addition, the therapeutic potential of delivering recom binant GCAP1, both directly and via liposomes, is explored as a strategy to modulate the phototransduction cascade in IRDs. This approach proves that targeted protein delivery can effectively alter photoreceptor responses in mice models, marking a promising avenue for treating retinal diseases. Through a comprehensive analysis, this thesis provides novel insights into the complex regulatory mechanisms of phototransduction and underscores the potential of innovative therapeutic strategies for IRDs.