RESUMO
Ichthyosis with confetti (IWC) is a genodermatosis associated with dominant-negative variants in keratin 10 (KRT10) or keratin 1 (KRT1). These frameshift variants result in extended aberrant proteins, localized to the nucleus rather than the cytoplasm. This mislocalization is thought to occur as a result of the altered carboxy (C)-terminus, from poly-glycine to either a poly-arginine or -alanine tail. Previous studies on the type of C-terminus and subcellular localization of the respective mutant protein are divergent. In order to fully elucidate the pathomechanism of IWC, a greater understanding is critical. This study aimed to establish the consequences for localization and intermediate filament formation of altered keratin 10 (K10) C-termini. To achieve this, plasmids expressing distinct KRT10 variants were generated. Sequences encoded all possible reading frames of the K10 C-terminus as well as a nonsense variant. A keratinocyte line was transfected with these plasmids. Additionally, gene editing was utilized to introduce frameshift variants in exon 6 and exon 7 at the endogenous KRT10 locus. Cellular localization of aberrant K10 was observed via immunofluorescence using various antibodies. In each setting, immunofluorescence analysis demonstrated aberrant nuclear localization of K10 featuring an arginine-rich C-terminus. However, this was not observed with K10 featuring an alanine-rich C-terminus. Instead, the protein displayed cytoplasmic localization, consistent with wild-type and truncated forms of K10. This study demonstrates that, of the various 3' frameshift variants of KRT10, exclusively arginine-rich C-termini lead to nuclear localization of K10.
Assuntos
Arginina/genética , Núcleo Celular/genética , Eritrodermia Ictiosiforme Congênita/genética , Queratina-10/genética , Mutação , Transporte Ativo do Núcleo Celular/genética , Alanina/genética , Alanina/metabolismo , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Éxons/genética , Mutação da Fase de Leitura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Queratina-10/química , Queratina-10/metabolismo , Queratinócitos/metabolismo , Microscopia ConfocalRESUMO
Blinding diseases of the retina are frequently characterized by loss of photoreceptor cells. The retinal degeneration 10 (rd10) mouse expresses a mutant form of rod phosphodiesterase leading to autosomal recessive photoreceptor degeneration. In contrast to rd1, rd10 mice have remaining rod function mimicking more closely most forms of human Retinitis Pigmentosa. Here we use morphology, biochemistry, retinal whole mounts, real-time PCR, Western blotting and immunofluorescence to compile a comprehensive report on progression of retinal degeneration in the rd10 retina up to one year of age. We show that retinal development, morphology, gene expression pattern and retinal vasculature was normal until postnatal day 15. Thereafter, a bi-phasic pattern of rod cell death emerged with a first rapid phase peaking around 3 weeks of age followed by a slower second phase. Death of cone cells followed with a delay and vessel dropout was prominent in the retinal periphery of 6 months old rd10 mice. At one year of age, RPE atrophy was evident. The degenerating retina rapidly induced expression of transcriptional regulators Atf3 and Cebpd. Induction of Atf3 was transient and lasted only for several days at the beginning of degeneration whereas levels of Cebpd remained elevated throughout the period of photoreceptor loss. Several protective genes such as Lif, Edn2 and Fgf2 which are implicated in a potent endogenous survival pathway, and Mt1 and Mt2 were strongly upregulated in the rd10 retina. In addition, increased expression of Casp1 and Il1b suggested an inflammatory response.
Assuntos
Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Degeneração Retiniana/genética , Transdução de Sinais/fisiologia , Fator 3 Ativador da Transcrição/genética , Animais , Animais Recém-Nascidos , Apoptose , Atrofia , Western Blotting , Proteína delta de Ligação ao Facilitador CCAAT/genética , Sobrevivência Celular/fisiologia , Progressão da Doença , Endotelina-2/genética , Fator 2 de Crescimento de Fibroblastos/genética , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Fator Inibidor de Leucemia/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Fotorreceptoras de Vertebrados/patologia , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Vasos Retinianos/patologiaRESUMO
A common feature of neurodegenerative disorders is acute or progressive loss of neurons due to apoptosis. The pathological isoform of the prion protein is associated with retinal apoptosis and the cellular isoform (PrPc) has been shown to mediate protection from apoptosis in cell culture and in neonatal retinal explants. Using a model of light-induced photoreceptor apoptosis, we show in vivo that the levels of PrPc expression in the retina inversely correlate with the susceptibility of photoreceptors to light damage. Dissection of apoptotic signalling cascades suggests that PrPc acts neuroprotectively downstream of AP-1 induction. Our results reveal PrP as a neuroprotective/anti-apoptotic factor in vivo and suggest that PrPc may function as a guardian of neuronal integrity.