Dromaretsʹkyy A. Perturbed Са2+ -dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia

Calcium (Ca2+) signaling plays an essential role in neuronal activity and regulation. Any disruption in this signaling can lead to neurological issues, ranging from minor disorders to severe diseases. The process of interpreting and converting Ca2+ signals into internal neuronal regulation is managed by various systems, including neuronal Ca2+ sensor proteins (NCS), which are key players in this process. The operation of these sensor proteins and their interactions with other systems can be complex, making it challenging to detect dysfunctions, let alone repair them. A deeper understanding of the molecular and cellular mechanisms underlying this proteins operation is essential to address these challenges and advance our ability to manage neurological disorders. Primary autosomal-recessive dystonia (DYT2) is a neurological movement disorder syndrome that results in sustained or repetitive muscle contractions. Such contractions cause twisting and repetitive movements or abnormal painful postures, negatively affecting the quality of life. Recent publications demonstrate the connection between this condition and mutations in the gene of neuronal calcium sensor protein hippocalcin (HPCA), namely missense mutations N75K and T71N. The gene is almost exclusively expressed in the brain with high level of expression in the cortex, striatum, cerebellum and hippocampus, i.e. in brain areas revealing abnormalities in dystonia. HPCA contains three EF-hand domains capable of binding Ca2+. The binding results in a Ca2+-myristoyl switch, a Ca2+-dependent conformation change leading to protrusion of its myristoyl-containing N-terminal region out of a hydrophobic pocket of the molecule. This allows HPCA to translocate from the cytosol to the plasma membrane. It is established that this Ca2+-dependent translocation of HPCA leads to inhibition of cortical and hippocampal neurons by gating a slow afterhyperpolarization (sAHP) current. The research aimed to find differences in biophysical properties of mutant variants N75K and T71N of the hippocalcin protein compared with a wild-type variant and resulting alterations in the function of molecular signaling to which this protein contributes. The research was conducted with the use of genetic, electrophysiological, and fluorescent methods. A special technique was developed to estimate the concentration of the expressed variants of hippocalcin with an attached fluorescent tags. The technique is based on the calculations involving optical spectral properties of the equipment and used fluorophores as well as other fluorescent dyes that served as references with known concentrations. This research demonstrates that mutation N75K, which was associated with dystonia DYT2, causes HPCA to lose its functions as a calcium ion sensor under conditions of physiological neural activity. Due to the altered capability of the mutated hippocalcin to perform Ca2+-dependent translocation to the plasma membrane, this variant of hippocalcin is unable to control slow after-hyperpolarization in neurons. Translocation of the N75K hippocalcin mutant to the plasma membrane of neuronal apical dendrites that is responsible for the generation of a sAHP current was severely reduced compared to the wild-type hippocalcin variant. This reduction led to the inability of the mutated hippocalcin variant N75K to generate additional sAHP current to one generated by the translocation of the endogenic wild-type hippocalcin. In contrast, the expression of wild-type hippocalcin caused that current to rise. Yet this mutation N75K does not affect the myristoyl switch, nor the spatial distribution of the hippocalcin binding to plasma membrane. Mutated hippocalcin variant T71N did not exhibit any significant changes in the translocation. In general, the hippocalcin mutation N75K causes increased neuronal excitability due to reduced sAHP and its inhibition effect in response to a series of action potentials bursts and θ-stimulation. It follows that the postsynaptic currents which occur subsequent to the action potential burst possess an increased likelihood of inducing further action potentials in the affected neurons. This phenomenon may serve as the fundamental cause of the motor symptoms observed in the dystonia DYT2 movement disorder.