# name
Genetic and biochemical approaches to decipher the molecular basis of the pathology in LCA12 and RP10 retinal dystrophies

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Universitat de Barcelona. Departament de Ciències Fisiològiques

# metadata.dc.contributor.other[].value
Universitat de Barcelona. Departament de Ciències Fisiològiques

# metadata.dc.description.abstract[].value
[eng] The main aim of this work was to characterize the molecular mechanisms that underlie the physiopathology of LCA12 and RP10, that are inherited retinal dystrophies caused by alterations in guanine nucleotide metabolism. First, LCA12 is caused by loss-of-function mutations in the retinal degeneration 3 (RD3) gene, that impair rod and cone function and cause a fast retinal degeneration in patients. This disease is reproduced in the natural strain of rd3 mice. The underlying physiopathology mechanisms of LCA12 are not well understood. We previously proposed that Guanylate Cyclase Activating Proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in six weeks in rd3 mice, it takes eight months to halve in rd3/rd3 GCAPs-/- mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segments in their Ca2+-free, guanylate- cyclase-activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum stress, mitochondrial swelling and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of LCA12 this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision. Second, RP10 is caused by gain-of-function mutations in IMPDH1 gene. Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells of the retina, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness, but the physiological relevance of IMPDH1 in the retina or its in vivo regulation remain largely unknown. We here investigate the in vivo regulation and physiological relevance of IMPDH1 in the retina, to gain insight into how IMPDH1 mutations lead to retinal dystrophies. We here report that retinal IMPDH1 in vivo is regulated by light- dependent phosphorylation at Thr159/Ser160 in the Bateman domain by protein kinase C, that desensitizes the enzyme to allosteric inhibition by GDP/GTP. The projected enhancement in guanine 15 nucleotide synthesis would contribute to sustain the GTP levels during illumination. Accordingly, we show that living mice accumulate IMPDH1 aggregates at rod outer segments upon prolonged bright light exposure; and that inhibiting IMPDH1 activity in living mice by intravitreal injection of IMPDH1 inhibitors delays rod mass response recovery. Based on these results we propose a novel mechanism of in vivo regulation of IMPDH1 that when disrupted leads to blindness.

# metadata.dc.source[].value
Tesis Doctorals - Departament - Ciències Fisiològiques

# metadata.dc.title[].value
Genetic and biochemical approaches to decipher the molecular basis of the pathology in LCA12 and RP10 retinal dystrophies