Alteraciones en la degradación intracelular de proteínas y en la endocitosis en las lipofuscinosis ceroideas neuronales infantil tardía y juvenil

Las lipofuscinosis ceroideas neuronales son un grupo heterogéneo de enfermedades neurodegenerativas hereditarias graves que tienen su inicio en la infancia y que se engloban dentro de las enfermedades de almacenamiento lisosomal. Las características clínicas de estas patologías son una disminución de la capacidad mental acompañada de alteraciones motoras, epilepsia y pérdida de visión. Histológicamente las células muestran una acumulación de material autofluorescente (lipofuscina) en el interior de los lisosomas de diversos tejidos, pero en especial en el cerebro y la retina. De entre todas las lipofuscinosis ceroideas neuronales, este trabajo se ha centrado en dos de ellas, la LINCL (ORPHA 168491) y la JNCL (ORPHA 79264), puesto que, conjuntamente, suponen aproximadamente el 90 % de todos los casos de lipofuscinosis ceroidenas neuronales que se diagnostican en el mundo. Estas dos variantes son consecuencia de mutaciones en los genes CLN2 (LINCL) y CLN3 (JNCL), que respectivamente codifican para una enzima lisosomal, la tripeptidil peptidasa I (TPP1), y para una proteína transmembrana, Cln3p, cuya función precisa actualmente se desconoce. No obstante, se postula que está asociada especialmente a microdominios de membrana enriquecidos en esfingolípidos y colesterol, localizados particularmente en el sistema endosomal/lisosomal. Pese a la heterogeneidad de las proteínas implicadas y a la diversidad de su localización subcelular, las similitudes entre los diferentes fenotipos de lipofuscinosis ceroideas neuronales sugieren que esas proteínas podrían jugar distintos papeles en una ruta común relacionada con los lisosomas y que sería especialmente importante en neuronas. La acumulación de material sin degradar en el interior de los lisosomas de las células de estos pacientes, que puede observarse mediante microscopía electrónica con una morfología diferente según cada variante (curvilíneas en LINCL y en forma de huella dactilar en JNCL), presenta como rasgo común elevadas cantidades de la subunidad c de la ATP sintasa mitocondrial. Esto indica que las proteínas codificadas por los genes CLN2 y CLN3 pueden estar implicadas o participar de forma directa o indirecta en la degradación de esa subunidad y que, como consecuencia de mutaciones en estos genes, se produciría esta acumulación que alteraría la actividad degradativa a través de las vías lisosomales, y en especial de la macroautofagia, puesto que esta es la principal vía lisosomal de degradación intracelular de proteínas. El objetivo principal de este proyecto ha sido investigar las bases celulares que pudieran explicar la diferente gravedad de las dos principales variantes de las lipofuscinosis ceroideas neuronales, la LINCL y la JNCL. Para ello, y puesto que ambas acumulan lipofuscina en el interior de los lisosomas, se ha realizado un análisis comparativo de similitudes y diferencias en alteraciones relacionadas con el funcionamiento normal de las principales vías de degradación intracelular de proteínas. Concretamente, hemos estudiado las posibles alteraciones en: 1. Los sistemas de degradación intracelular de proteínas, especialmente la autofagia y las vías de señalización que la regulan. 2. El tráfico vesicular, tanto en las vías endocíticas como en el transporte de enzimas lisosomales. Para estudiar las alteraciones en los sistemas de degradación intracelular de proteínas y en las vías de señalización que la regulan (Objetivo 1), hemos utilizado las siguientes aproximaciones: a) el estudio comparativo de la estructura de los lisosomas en las células de estos pacientes a través de la microscopía electrónica convencional; b) la determinación mediante un ensayo in vitro de la actividad de la enzima TPPI, mutada en la variante LINCL; c) la medida de la actividad de las diferentes vías degradativas lisosomales, estudiando la degradación de las proteínas de vida media corta y larga utilizando valina marcada radiactivamente; d) el análisis de la síntesis y maduración de los autofagosomas mediante Western-blot a través de un marcador específico de esta vía degradativa, LC3-II; e) el estudio del pH lisosomal mediante microscopía de fluorescencia, analizando la incorporación a los lisosomas de FITC-dextrano; f) el estudio de la ubicuitinación mediante Western-blot y la actividad de los proteasomas mediante un ensayo in vitro de quimioluminiscencia y un experimento de "pulso" y "caza", puesto que es la principal vía no lisosomal de degradación intracelular de proteínas, y podría modular su actividad ante defectos en las vías lisosomales; g) los niveles de ROS y sus consecuencias en el ciclo celular, ambos, mediante citometría de flujo; h) los niveles de fosforilación mediante Western-blot de diversas proteínas implicadas en la regulación por hormonas y nutrientes de la macroautofagia, como Akt, ERK1/2, p38α y de dos sustratos de la quinasa mTOR que ocupa un puesto central en esta regulación, p70 S6K y 4EBP1. Todos estos ensayos se han realizado tanto en situación de ayuno, en la que se activa la macroautofagia, como añadiendo insulina y aminoácidos (hormona y nutrientes) para disminuir esta actividad. Para estudiar las alteraciones en el tráfico vesicular (Objetivo 2), hemos utilizado las siguientes aproximaciones: a) el estudio de la macropinocitosis a través del análisis mediante citometría de flujo de la cinética de incorporación de FITC-dextrano; b) el estudio de la endocitosis mediada por receptor, analizando de forma separada los procesos de fosforilación, internalización, reciclaje y degradación del receptor de crecimiento epidérmico, utilizando técnicas de citometría de flujo y Western-blot; c) el estudio del transporte de las enzimas lisosomales desde la red trans del Golgi mediante el receptor de manosa-6-fosfato independiente de cationes, analizando la estabilidad de los componentes de dicho receptor mediante Western-blot; d) el estudio del reciclaje del receptor hacia la red trans del Golgi por parte del retrómero, analizando la estabilidad de sus componentes mediante Western-blot, así como la colocalización del receptor de manosa-6-fosfato independiente de cationes, y la red trans del Golgi mediante experimentos de colocalización, utilizando la microscopía de fluorescencia con anticuerpos específicos frente a estos componentes. Las conclusiones de este trabajo son: De las dos principales vías de degradación intracelular de proteínas, solo la macroautofagia es defectuosa en las líneas celulares de pacientes con lipofuscinosis ceroideas neuronales infantil tardía (CLN2) y juvenil (CLN3). En cuanto a las principales vías endocíticas estudiadas, solo la macropinocitosis está reducida en ambas líneas de pacientes. En los fibroblastos de pacientes se produce una menor formación de autofagosomas que coincide con una mayor activación de la vía Akt-mTOR, especialmente en los fibroblastos CLN2. En los fibroblastos CLN3, el pH lisosomal es mayor en aproximadamente 0,5 unidades que en los fibroblastos control o CLN2. Como consecuencia de esto, la maduración de autofagosomas, el transporte vesicular y la maduración de las enzimas lisosomales están disminuidos en aquellas células. Asimismo, la actividad de la enzima TPP1, cuyo pH óptimo es muy ácido, disminuye en aproximadamente un 60 % en los fibroblastos CLN3 en comparación con los controles. En los fibroblastos CLN2 están más activadas las quinasas p38α y ERK1/2, son mayores los niveles de ROS y es menor la actividad de la catalasa en comparación con los fibroblastos control o CLN3. Esto, junto a la prácticamente total ausencia de la actividad de TPP1, podría explicar la aparición más temprana de la sintomatología en la variante infantil tardía

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

Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages

Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE-/-) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE-/- macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress

Alterations in ROS Activity and Lysosomal pH Account for Distinct Patterns of Macroautophagy in LINCL and JNCL Fibroblasts

<div><p>Neuronal Ceroid Lipofuscinoses (NCL) are lysosomal storage disorders characterized by the accumulation of lipofuscin within lysosomes. Late infantile (LINCL) and juvenile (JNCL) are their most common forms and are caused by loss-of-function mutations in tripeptidyl peptidase 1 (TPP1), a lysosomal endopeptidase, and CLN3 protein (CLN3p), whose location and function is still controversial. LINCL patients suffer more severely from NCL consequences than JNCL patients, in spite of having in common an abnormal accumulation of material with a similar composition in the lysosomes. To identify distinctive characteristics that could explain the differences in the severity of LINCL and JNCL pathologies, we compared the protein degradation mechanisms in patientś fibroblasts. Pulse-chase experiments show a significant decrease in protein degradation by macroautophagy in fibroblasts bearing TPP1 (CLN2) and CLN3p (CLN3) mutations. In CLN2 fibroblasts, LC3-II levels and other procedures indicate an impaired formation of autophagosomes, which confirms the pulse-chase experiments. This defect is linked to an accumulation of Reactive Oxygen Species (ROS), an upregulation of the Akt-mTOR signalling pathway and increased activities of the p38α and ERK1/2 MAPKs. In CLN3 fibroblasts, LC3-II analysis indicates impairment in autophagosome maturation and there is also a defect in fluid phase endocytosis, two alterations that can be related to an observed increase of 0.5 units in lysosomal pH. CLN3 fibroblasts also accumulate ROS but to a lower extent than CLN2. TPP1 activity is completely abrogated in CLN2 and partially diminished in CLN3 fibroblasts. TPP1 cleaves small hydrophobic proteins like subunit c of mitochondrial ATP synthase and the lack or a lower activity of this enzyme can contribute to lipofuscin accumulation. These alterations in TPP1 activity lead to an increased ROS production, especially in CLN2 in which it is aggravated by a decrease in catalase activity. This could explain the earlier appearance of the symptoms in the LINCL form.</p> </div

Lysosomal alterations in NCL fibroblasts.

<p>(A) Representative electron micrographs of control, CLN2 and CLN3 fibroblasts incubated in high proteolysis medium to activate autophagy. Arrows indicate lysosomal vacuoles. Bar: 0.5 µm. (B) TPP1 activity measurements. Control (black bars), CLN2 (white bars) and CLN3 (gray bars) fibroblasts were incubated under high (H) and low (L) proteolysis conditions as indicated. TPP1 activity and total protein were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results are expressed as RFU values per minute and µg protein, and are the mean and S.D. from three separate experiments with triplicated samples. (C) Pulse-chase experiments. Labelling of human fibroblasts with [³H] valine in high proteolysis medium, measurements of degradation of long-lived proteins and the contribution of lysosomal (total and macroautophagy) and proteasomal pathways were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results are presented as the percentage of the labelled protein that is degraded per hour, and are the mean and S.D. from eight to fifteen separate experiments with duplicated samples. In B and C, stars immediately on top of bars indicate statistically significant differences from control values (*p<0.05 and ***p<0.005).</p

Lysosomal pH values of control, CLN2 and CLN3 fibroblasts incubated in high and low proteolysis media.

<p>The mean fluorescence intensity values for each cell and condition, obtained in three separate experiments with duplicated samples, were interpolated in the corresponding calibration curves (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#pone.0055526.s004" target="_blank">Figure S4</a>). Stars indicate statistically significant differences from control values (**p<0.01).</p

Macropinocytosis is reduced in CLN2 and especially in CLN3 fibroblasts.

<p>(A) Representative fluorescence microscopic images of control, CLN2 and CLN3 fibroblasts incubated at 37°C for 2 h with 0.5 mg/ml FITC-dextran, followed by a 30 min chase. Bar: 50 µm. Insets show cells at higher magnification. Bar: 150 µm. (B) To quantify endocytic internalization, control, CLN2 and CLN3 fibroblasts were incubated at 37°C for the indicated periods of time with 0.5 mg/ml FITC-dextran, washed and analyzed by flow citometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results indicate the percentage of fluorescently labelled cells, and are the mean and S.D. from three separate experiments with triplicated samples. Representative dot blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#pone.0055526.s003" target="_blank">Figure S3A</a>. Stars indicate statistically significant differences from the corresponding control values at each time (*p<0.05, **p<0.01 and ***p<0.005).</p

The Akt-mTOR pathway is more activated in CLN3 and especially in CLN2 fibroblasts.

<p>(A) Control (CTRL), CLN2 and CLN3 fibroblasts were incubated in high (H) and low (L) proteolysis media. Lysates were analyzed by western blot using antibodies against the phosphorylated forms of various proteins upstream and downstream of mTOR: Akt-Thr 308 (P-Akt-T), Akt-Ser 473 (P-Akt-S), p-38α (P-p38α), ERK 1/2 (P-ERK 1/2), p70S6K (P-p70S6K) and 4EBP1 (P-4EBP1) and their respective total proteins. The bands corresponding to the phosphorylated proteins, obtained from three different experiments, were densitometred and normalized to the corresponding bands of total protein. Data are plotted in percentage relative to the highest value. Stars indicate statistically significant differences from control values under high and low proteolysis conditions (*p<0.05, **p<0.01 and ***p<0.005). (B) Control, CLN2 and CLN3 fibroblasts were incubated for the indicated times in high proteolysis medium with EGF (100 ng/ml). Total extracts were analyzed using P-Akt-T and total Akt antibodies. A representative blot is shown. The bands corresponding to phosphorylated and total Akt were quantified by densitometry as in A). Stars indicate statistically significant differences from control values at each time (***p<0.005).</p

Reduced autophagosome formation in CLN2 and CLN3 fibroblasts and decreased autophagosome maturation in CLN3 cells.

<p>(A) Total extracts from control (CTRL), CLN2 and CLN3 fibroblasts incubated under high (H) and low (L) proteolysis conditions and with or without lysosomal inhibitors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Material and Methods</a>) were analyzed by western-blot using LC3 and, as a loading control, actin antibodies. The position of LC3-I, and LC3-II bands are indicated on the left. Densitometric measurements from three different experiments are shown below. Results are presented as LC3-II/actin ratios and are expressed as percentages of the control values with inhibitors under high proteolysis conditions. Stars indicate statistically significant differences under high proteolysis conditions from the corresponding control values (**p<0.01). (B) Representative fluorescence images of control, CLN2 and CLN3 fibroblasts incubated in high proteolysis medium for 2 h at 37°C. Upper micrographs: immunocytochemistry with anti-LAMP1. Lower micrographs: control, CLN2 and CLN3 fibroblasts, 48 h after transfection with pEGFP-LC3. Bar: 20 µm.</p

Lysosomes in CLN3 fibroblasts have a higher pH.

<p>(A) Exponentially growing control, CLN2 and CLN3 fibroblasts were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Cells were observed by fluorescence microscopy using an inverted microscope and over 200 images were randomly taken at each pH. Upper and lower images show representative photographs of, respectively, permeabilized control fibroblasts incubated in media at pHs 4.5 (left) and 6.5 (right), and non-permeabilized control (left) and CLN3 (right) fibroblasts incubated in high proteolysis medium (pH 7.4). Bar: 20 µm. (B) Effects of different pHs on TPP1 activity. Control fibroblasts were incubated under high proteolysis conditions and TPP1 activity was measured at different pHs using AAF-AMC. Measurements of AMC fluorescence and quantification of protein were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055526#s4" target="_blank">Materials and Methods</a>. Results are expressed as RFU values per minute and µg protein and are the mean and S.D. from three separate experiments with triplicated samples. Stars indicate statistically significant differences from pHs 4.0 and 4.5 (***p<0.005).</p