Alterations in EGF-Endocytosis, Lysosomal Enzyme Transport and Maturation of Cathepsins in Juvenile Neuronal Ceroid Lipofuscinosis Fibroblasts

Background: Juvenile neuronal ceroid lipofuscinosis (JNCL), one of the most frequent forms of the NCL storage diseases, is known to be caused by loss-of-function mutations in ceroid-lipofuscinosis neuronal protein 3 (CLN3), but its cell function has not been fully elucidated. We previously reported increased lysosomal pH in CLN3 deficient cells. In the present study, we analysed the consequences of this effect in the endo-lysosomal pathways in CLN3 cells.Methods: The present study investigated different endo-lysosomal pathways in control, CLN2, CLN3 human skin fibroblasts under high and low proteolysis conditions. Cell surface biotinylation after EGF (2 ng/mL) stimulation, EGF phosphorylation (Tyr-845), retromer and cation-independent mannose-6-phosphate receptor (CI-MPR) levels and stability, EGF degradation pathways and cathepsin L and D levels were analysed by western blots. Caveolae mediated endocytosis was analysed by flow cytometry. CIMPR subcellular localization was ascertained by immunocytochemistry, confocal microscopy and further image analysis.Results: Whereas caveolae-mediated endocytosis was not affected in CLN3 cells, clathrin-mediated epidermal growth factor (EGF) internalization was reduced, along with EGF receptor (EGFR) phosphorylation. In addition, cell surface EGFR levels and recycling to the cell membrane were increased. EGFR lysosomal degradation was impaired and our results suggest that the receptor was diverted to proteasomal degradation. We also analysed the machinery responsible for lysosomal hydrolase transport to the lysosome and found increased stability of CIMPR, a major receptor implicated in the transport of hydrolases. The subcellular distribution of the CI-MPR was also altered in CLN3 cells, since it accumulated within the Trans-Golgi network (TGN) and did not progress into the lysosomes. In addition, we found a reduced turnover of retromer subunits, a complex that retrieves the CI-MPR from endosomes to the TGN. Finally and as a possible consequence of these alterations in lysosomal enzyme transport, cathepsin L and D maturation were found suppressed in CLN3 cells.Conclusion: Altogether, these results point to increased lisosomal pH as a pivotal event causing various alterations in intracellular traffic associated to the development of JNCL disease

Neurodegeneration and Epilepsy in a Zebrafish Model of CLN3 Disease (Batten Disease)

The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80–85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery

Expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry

<p>Abstract</p> <p>Background</p> <p>The number of breast cancer patients diagnosed with ductal carcinoma <it>in situ </it>(DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences.</p> <p>Methods</p> <p>For detection of TACA expression, specimens were stained with <it>Griffonia simplicifolia </it>lectin-I (GS-I) and <it>Vicia vilosa </it>agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors.</p> <p>Results</p> <p>We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; <it>P </it>= 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; <it>P </it>= 0.0004).</p> <p>Conclusion</p> <p>The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk.</p

Utilidad de los valores de troponina y su relación con la función renal a su llegada, en la evaluación en Urgencias de los pacientes con dolor torácico de origen incierto y primera determinación de troponina normal

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2021/2022Actualmente la estratificación de los pacientes con dolor torácico agudo de origen incierto y troponina (Tn) normal sigue siendo un reto. El objetivo de nuestro trabajo fue estudiar el valor pronóstico añadido de la función renal sobre el que ya de por sí tienen los datos clínicos y los niveles de troponina cardíaca de alta sensibilidad (TnC-as). Se analizó de forma retrospectiva una muestra de 2254 pacientes consecutivos que acudieron a urgencias por presentar un episodio de dolor torácico agudo de origen incierto con primera determinación de TnC-as normal y en los que también se determinó la función renal a su llegada. Se realizó un seguimiento de la evolución clínica, recogiendo los eventos de muerte, infarto agudo de miocardio y revascularización hasta 1 año tras el alta. El análisis estadístico de los datos recogidos mostró que el valor pronóstico de la combinación de la TnC-as y la función renal junto con la escala clínica de riesgo HEART (ABC 0,871) fue el modelo predictivo que mejores resultados ofrecía, por encima de la TnC-as en la primera determinación (ABC 0,783), de la función renal (ABC 0,565) y de las escalas clínicas de riesgo de forma aislada (GRACE 0,692; TIMI 0,794; HEART 0,843). La combinación de los niveles de TnC-as en la primera determinación y de la función renal, junto con las escalas clínicas de riesgo, presentó la mejor capacidad discriminativa y predictora en la estratificación de riesgo a 1 año.niveles de troponina cardíaca de alta sensibilidad (TnC-as). Se analizó de forma retrospectiva una muestra de 2254 pacientes consecutivos que acudieron a urgencias por presentar un episodio de dolor torácico agudo de origen incierto con primera determinación de TnC-as normal y en los que también se determinó la función renal a su llegada. Se realizó un seguimiento de la evolución clínica, recogiendo los eventos de muerte, infarto agudo de miocardio y revascularización hasta 1 año tras el alta. El análisis estadístico de los datos recogidos mostró que el valor pronóstico de la combinación de la TnC-as y la función renal junto con la escala clínica de riesgo HEART (ABC 0,871) fue el modelo predictivo que mejores resultados ofrecía, por encima de la TnC-as en la primera determinación (ABC 0,783), de la función renal (ABC 0,565) y de las escalas clínicas de riesgo de forma aislada (GRACE 0,692; TIMI 0,794; HEART 0,843). La combinación de los niveles de TnC-as en la primera determinación y de la función renal, junto con las escalas clínicas de riesgo, presentó la mejor capacidad discriminativa y predictora en la estratificación de riesgo a 1 año.Currently, the stratification of patients with acute chest pain of uncertain origin and normal troponin (Tn) remains a challenge. Our objective was to study the prognostic value that the determination of renal function adds over the value of clinical data and high-sensitivity cardiac troponin (hs-cTn) levels alone. We retrospectively included 2,254 consecutive patients who presented to our emergency department with acute chest pain of uncertain origin and normal hs-cTn in the first determination and in whom renal function was also determined upon arrival. Follow-up of was carried out, collecting the combined endpoint of death, acute myocardial infarct and revascularization up to 1 year after discharge. The statistical analysis of the collected data showed that the prognostic value of the combination of hs-cTn and renal function together with the HEART clinical risk scale (AUC 0.871) was the predictive model that offered the best results, above TnC -as in the first determination (AUC 0.783), renal function (AUC 0.565) and clinical risk scores (GRACE 0.692; TIMI 0.794; HEART 0.843) alone. The combination of hs-TnC levels in the first determination and renal function together with the clinical risk scales, presented the best discriminative and predictive capacity in risk stratification at 1 year