Biology subjects

Biasi, A.

Publications and source records attributed to Biasi, A..

3 recordsLinked to original sources

bioRxiv2026

A knock-in model of severe GUCA1A cone-rod dystrophy reveals retinal network dysfunction beyond phototransduction

Autosomal dominant cone-rod dystrophy caused by GUCA1A mutations is generally viewed as a disorder of phototransduction, yet the mechanisms linking photoreceptor dysfunction to progressive vision loss remain unclear. Here, using a knock-in mouse carrying the severe GCAP1 p.(E111V) variant, we show that retinal network dysfunction precedes structural degeneration. Mutant mice exhibited delayed rod photoresponses, increased light sensitivity, selective visuospatial deficits, and progressive impairment of visually evoked responses in the superior colliculus and visual cortex, demonstrating propagation of functional deficits beyond photoreceptors. Transcriptomic and ultrastructural analyses revealed early synaptic, mitochondrial and inflammatory alterations despite largely preserved retinal architecture. Acute ex vivo delivery of recombinant wild-type GCAP1 partially restored mutant rod photoresponse kinetics, indicating that these early functional deficits remain biochemically modifiable. These findings redefine severe GUCA1A-associated disease as a progressive disorder of retinal network function, identifying an early therapeutic window before structural degeneration. One-Sentence SummaryVisual function breaks down long before photoreceptors are lost, opening an early window for intervention.

neuroscience↗

bioRxiv2024

Supramolecular complexes of GCAP1: towards the development of effective biologics for inherited retinal dystrophies

Guanylate Cyclase Activating Protein 1 (GCAP1) is a neuronal Ca2+-sensor protein expressed in photoreceptors where it regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in a Ca2+-dependent manner. Recently, over 20 missense mutations in GUCA1A (encoding for GCAP1) have been associated with inherited autosomal dominant retinal diseases, namely cone dystrophy (COD) and cone-rod dystrophy (CORD). Since GCAP1 is known to be a functional dimer, COD/CORD patients present a heterogeneous pool of GCAP1 assemblies constituted by wild-type and mutated homodimers and heterodimers. Here, we present an integrated in silico and biochemical investigation on the effects of the E111V substitution, associated with a severe form of CORD, on GCAP1 homo- and hetero-dimerization. Despite inducing a constitutive activation of GC1 due to impaired Ca2+-binding in the high-affinity EF-hand 3 motif, the E111V substitution did not affect either the homo- or the hetero dimerization process as clearly highlighted by aSEC and molecular docking experiments. Indeed, both variants exhibited micromolar monomer-dimer equilibrium constants in the presence of both Mg2+ and Ca2++, indicating that at physiological cellular concentrations both variants are predominantly monomers under Ca2+-loaded and, to a lesser extent, Mg2+-loaded conditions. Molecular docking and dynamics simulations confirmed chromatographic results highlighting slight alterations in free energy of binding involving the pathogenic E111V variant in the Ca2+-bound state and increased mobility over time affecting the Ca2+-coordinating EF3 motif. In addition, to evaluate possible therapeutic approaches, the regulation of the catalytic activity of GC1 by WT and E111V-GCAP1 was studied in the presence of retinal degeneration protein 3 (RD3), an -helical protein that strongly inhibits GC1, and a RD3-derived peptide (RD3ppt) which encompasses a region of RD3 that is essential for its inhibitory activity. GC1 activity assays in the presence of RD3ppt suggest that the enzymatic activity is partially inhibited by the peptide at low micromolar concentrations when GCAP1 variants are present. The incomplete shut down of GC1 by RD3 could be explained by the interaction occurring between RD3 and GCAP1, known to form a complex with GC1 in the endoplasmic reticulum. This fundamental interaction was here investigated spectroscopically and in silico, unveiling major structural rearrangements upon complex formation. Interestingly, the full RD3 protein was able to better modulate GC1 activity and restore the abnormal cGMP production induced by the pathogenic E111V-GCAP1 variant to a physiological level.

biochemistry↗

bioRxiv2022

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina

Retinal dystrophies of genetic origin are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). Analysis of protein biodistribution and direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

neuroscience↗