Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations.
Lysosomes are acidic intracellular vesicles that provide an optimal physicochemical milieu for enzymatic activities, most of them catabolic, which need to be controlled. A well-recognized lysosomal function is the degradation and recycling of defective cellular material through autophagy, and of extracellular material that reach lysosomes by endocytosis or phagocytosis. Newly documented functions such as energy and nutrient sensing, secretion, plasma membrane repair, immune response and cell death, reveal lysosomes as sophisticated organelles controlling fine decisions in the life of a cell [reviewed by 1,2, 3]. Lysosome function is essential for human health, as clearly shown by the existence of numerous Lysosomal Storage Diseases [reviewed by 4], inherited metabolic disorders that affect a variety of tissues and organs and are particularly devastating for the nervous system. Moreover, lysosomal dysfunction is known to underlie the pathogenic mechanisms of neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as physiological aging [5,6, 7]. In the nervous system, proper lysosomal and autophagic functions are essential for the survival of postmitotic neurons, for the phagocytic activity of microglia and for the myelination process performed by Schwann cells and oligodendrocytes [8,9, 10,11]. The stability of lysosomal membrane is a key factor determining cell survival-death signaling [12], and its composition is essential for an efficient lysosomal enzymatic activity [13]. Reactive oxygen species (ROS) compromise lysosomal integrity due to membrane lipid peroxidation [12,14]. Because of the critical role of its luminal pH, the alkalinization of lysosomes due to proton leakage is thought to contribute to many pathologies [15], and compensatory responses or therapeutic manipulations that restore lysosomal pH would result in clear benefits [16]. Interestingly, not all lysosomes are equally sensitive to oxidative stress or have the same luminal pH [14,17]. Apolipoprotein D (ApoD), a lipid binding protein of the Lipocalin family first known as part of bloodstream lipoprotein particles, is one of the few genes consistently over-expressed in the aging brain, and in all neurodegenerative and psychiatric diseases tested so far [reviewed in 18]. At the biochemical level, ApoD lipid binding properties are well known [19,20,21,22,23,24] and its ability to inhibit lipid peroxidation by reducing radical-propagating lipid hydroperoxides has been determined [25]. At the functional level, evidence for cellular protection and pro-survival roles for ApoD keep accumulating both, in animal models and cell systems. ApoD exhibits a functional pleiotropy, connecting nervous system response to oxidative stress [26,27,28,29,30], recovery after injury [31,32], brain aging [33,34,35], or diverse forms of neurodegeneration [36,37,38], as well as longevity regulation [39,40,41,42,43] or nervous system control of the metabolic adaptations to stress [44]. For instance, a lack of ApoD in mice compromise the process of myelin phagocytosis after peripheral nervous system injury [31,32], and the autophagic process was revealed as a key element in the degeneration rescuing activity of the ApoD Drosophila melanogaster homolog Glial Lazarillo (GLaz) [37]. How can an extracellular lipid binding protein accomplish these intracellular functions? ApoD is internalized by different cell types including astrocytes and neurons [26,28,45,46]. However, the ApoD-containing intracellular organelles or the dynamics and route of entry have not been identified. To achieve a mechanistic knowledge of ApoD neuroprotective actions, an in-depth analysis of its subcellular traffic is required. In this work, we perform a comprehensive analysis of ApoD subcellular traffic and discover its role in lysosomal pH homeostasis and membrane stabilization under oxidative stress conditions. We demonstrate that ApoD is endocytosed and targeted to lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and actively helps to maintain lysosomal pH gradients both in astrocytes and in neurons, by maintaining lysosomal membrane integrity. Our analysis reveals ApoD as a specific marker for the subpopulation of lysosomes vulnerable to oxidation. We also test the notion that this lysosomal protecting mechanism is of wide biological consequences, by proving the role for this Lipocalin in two lysosomal-dependent biological processes such as myelin phagocytosis and optimization of neurodegeneration-triggered autophagy.
Using the Drosophila retina as a model system to assay neurodegeneration, we previously described that Type I Spinocerebellar Ataxia (SCA1) concurs with autophagic stress, showing an excessive or imbalanced induction of autophagy where autophagosome turnover is unable to keep pace with its formation [37]. GLaz, a Drosophila homologue of ApoD expressed by subsets of glial cells in the fly nervous system, has epistatic relationship with autophagy genes and optimizes clearance of aggregation-prone proteins such as the polyglutaminated form of human Ataxin 1 that is responsible for the SCA1 phenotype [37]. We concluded that GLaz rescues neurodegeneration by making autophagy more efficient, thus minimizing the negative effects of autophagic stress. We also proposed that the Lipocalin-mediated control of lipid peroxide levels influences autophagy at several steps, slowing down the process and ultimately making it more efficient. However, the mechanism for such an optimization of autophagy was not completely discerned. Here we evaluate retinal degeneration in the SCA1 fly retina model (Fig 1I) using FLEYE, a method for unbiased quantification based on the acquisition of fly eye surface pictures [47]. We combined the expression of polyglutaminated human Ataxin 1 (hATXN182Q) with GLaz, and with DorRNAi, a knock-down of the HOPS complex subunit Vps18/Dor critical for tethering and fusing autophagosomes with lysosomes [48] (Fig 1). GLaz neurodegeneration rescue (Fig 1C and 1D, compare with controls in Fig 1A and 1B) is completely abolished by DorRNAi expression (Fig 1E and 1F). Dor down-regulation itself does not produce significant neurodegeneration in normal conditions (Fig 1G) nor modifies the neurodegeneration phenotype triggered by SCA1 in the fly retina (Fig 1H). These results demonstrate that the lysosome-autophagosome fusion event is required for GLaz optimization of autophagy that finally results in an efficient clearance of misfolded proteins in neurodegenerative conditions in vivo. These results predict that Lipocalins must exert this function from the lysosome itself. Thus, a study showing the subcellular localization of ApoD-related Lipocalins once internalized in damaged neurons was required. To study the subcellular traffic of ApoD we switched to its native cell type, astrocytes, where we have reported its protective effect upon oxidative stress challenge, and its transcriptional regulation by the stress-responsive JNK pathway [26]. We studied ApoD subcellular localization by detecting the native protein in astroglial cells (1321N1 astroglioma cell line), avoiding transfections that could alter its physiological traffic. A set of nine different markers for intracellular organelles were used to evaluate colocalization with ApoD in control, low serum (LS) and treatment with the oxidative stress-inducing agent paraquat (PQ) at 2 and 24 hours after stimulus. Fig 2 and S1 Fig summarize the results of our image analysis from confocal z-stacks of at least 20 cells per condition, selected randomly from two independent experiments with triplicate wells (see Methods). Following two rounds of principal component analysis (PCA), we selected the intensity correlation quotient (ICQ) index [49] referenced to ApoD signal (ApoD ICQ) and the % Pixel Overlap referenced to ApoD signal (ApoD Overlap) to quantify ApoD protein targeted to each organelle (S1 Fig) (see Methods). We use a 2xICQ threshold of 0. 1 for a colocalization not to be considered due to chance [50]. Since most variables covariate between control and LS, only PQ referred to the control condition is shown for simplicity in most figures. ApoD concentrates significantly in Clathrin, EEA1, Lamp-2 and LC3-positive organelles, with particular prominence in the late endosome-lysosome compartment (LELC) positive for Lamp-2 (Fig 2A–2G). Borderline average values of 2xICQ are detected for ApoD colocalization with Caveolin-1 (Fig 2G), though some cells in the sample studied show clear colocalization over the threshold (Fig 2B). A significant PQ-dependent enrichment is observed in ApoD colocalization with Clathrin, Lamp-2 and LC3 at 24h of treatment (asterisks in Fig 2G and S2A–S2E Fig). At shorter times (2h) no PQ-dependent enrichment of ApoD is observed in the lysosomal or autophagosomal compartments of 1321N1 cells, while a prominent colocalization is seen for ApoD-Clathrin (asterisk in Fig 2H). In opposition, ApoD is not detected in mitochondria or peroxisomes, two organelles involved in oxidative stress generation and management (Fig 2G and S2F–S2G Fig). As expected for a Lipocalin, 1321N1 cells secrete ApoD to the culture medium (S3A Fig). Also, in ApoD-transfected human embryonic kidney-293T (HEK293T) cells, extracellular ApoD is detected with a stable accumulation in the culture medium over time (S3B Fig). However, no significant colocalization was observed with rough endoplasmic reticulum (RER; S3C Fig) and only borderline values were obtained with the Golgi apparatus (Fig 1A and 1G) of 1321N1 cells. A rapid passage through RER and Golgi in astroglial cells might render ApoD concentration below detection levels in those organelles, a hypothesis supported by the evident ApoD-RER colocalization obtained when ApoD is overexpressed in HEK293T cells (S3D Fig). To further analyze the spatial domain of ApoD overlap with organelle markers we calculated a percent pixel overlap (referenced to the ApoD signal) as a parameter independent of the fluorescence intensity taken into account in ICQ (Fig 2I). The representation of Clathrin pits, early endosomes, LELC and autophagosomes within the ApoD spatial domain is quite high (18–30%), indicating that these organelles are common residence sites for ApoD. No enrichment in spatial overlap is detected upon PQ treatment, suggesting that the elevated ApoD 2xICQ values reported above (Fig 2G and 2H) represent a stress- and time-dependent increase in ApoD concentration in those stable spatial domains. Given the prominent colocalization of ApoD with the LELC marker Lamp-2 in 1321N1 cells, we performed a more detailed time-course analysis of their spatial overlap. PQ triggers specifically a significant and transient increase in pixel overlap (Fig 3A), which is accompanied by a change in the distribution of the ApoD signal (Fig 3B): an initial phase of large and less numerous ApoD objects is followed by a late phase with more objects of small average size. This analysis suggests a PQ-dependent early enrichment of ApoD in LELC that might coincide with organelle fusion, probably autophagolysosomes, followed by ApoD traffic to smaller vesicles. Our colocalization studies, performed in conditions of membrane permeabilization (see Methods), uncovered that ApoD signal was also present in a peripheral dotted pattern after 24h of PQ treatment (Fig 3C, arrow in left panel). By performing ApoD immunolocalization in non-permeabilized conditions, we confirmed that this labeling is due to ApoD presence at the extracellular side of the plasma membrane (Fig 3C, arrowheads in right panel). Our object-based analysis of the plasma membrane located ApoD signal at 2 and 24h of PQ treatment (Fig 3D) shows two phases, which might be indicative of different cell biological processes: (i) An initial phase with increased number and size of objects is concordant with the early enrichment of ApoD in Clathrin-positive organelles (Fig 2H). (ii) Small, but still abundant, membrane-associated ApoD aggregates at 24h might point to a late traffic back to the membrane from the LELC. The early distribution of ApoD signal in permeabilized cells upon PQ treatment (Fig 2B) suggests organelle fusion, and ApoD is a common resident in LC3-positive organelles of 1321N1 cells (Fig 2F). Therefore, we studied ApoD traffic in the autophagy process. As predicted by the Drosophila in vivo results, we show that ApoD entry in autophagolysosomes is dependent on a proper lysosome-autophagosome fusion since chloroquine (CQ), known to alkalinize lysosomal pH and prevent its fusion [51], completely abolished colocalization of ApoD with LC3 (Fig 3E). We then quantified the colocalization of ApoD with Lamp-2 or LC3 (2xICQ index referenced to ApoD signal; Fig 3F). The presence of ApoD in LELC is relatively stable, while colocalization with LC3 drops below random levels both, when lysosome-phagosome fusion is impaired by CQ or when autophagy initiation is blocked at an early step using 3-methyladenine (3-MA). Likewise, a significant increase in ApoD-LC3 colocalization is observed when autophagy is stimulated by the mTOR inhibitor Rapamycin (Rap). An object-based analysis of ApoD-Lamp2-LC3 triple colocalization in 1321N1 cells exposed to PQ (Fig 3G and 3H), shows a transient increase in the volume of objects labeled by the three markers (ApoD-positive autophagolysosomes) and a decrease in their number. This suggests that a basal level of autophagic activity exists in these cells, and that after an initial phase of fusions of autophagolysosomes upon PQ treatment, autophagy is resolved with a net decrease of objects with the three markers. These data, together with the increased ApoD-LC3 colocalization after 24h PQ (Fig 2G) and a net PQ-dependent decrease in the number of LC3-positive vesicles (S4 Fig) suggest that ApoD is enriched in autophagolysosomes upon oxidative challenge. The subcellular localization of ApoD in 1321N1 cells in control and PQ conditions was further confirmed by morphological criteria of immunoelectron microscopy (Fig 4). In control conditions ApoD was mostly detected in early endosomes, small and located close to the plasma membrane (Fig 4A), as well as in lysosomes, electron- dense vesicles close to the nucleus (Fig 4B). Clear differences are observed upon PQ treatment in the representation of labeled subcellular localizations. After 2h of PQ treatment ApoD signal was observed on the plasma membrane (Fig 4C), being endocytosed in Clathrin-coated pits (Fig 4D), in larger late endosomes (Fig 4E), electron-dense lysosomes (Fig 4F), secondary lysosomes (Fig 4G), and autophagolysosomes (Fig 4H). In most cases, ApoD signal is observed associated to membranes. Interestingly, lysosomes under oxidative stress show an enrichment of membrane-associated ApoD (Fig 4F) compared to control conditions (Fig 4B). Since the LELC and autophagolysosomal compartments participate in protein degradation, we tested whether the ApoD passage through them simply reflects its degradation pathway. We used cells not expressing ApoD (HEK293T) subjected to a 2h pulse-chase experiments with purified human ApoD [39]. After a 2h period of ApoD exposure, ApoD labeling was evaluated in cells for up to 48h. The intracellular content of ApoD was very stable along the experimental period when we used native human ApoD (Fig 5A). However, a bacterially produced non-glycosylated human ApoD is rapidly endocytosed and quickly degraded (Fig 5B). Colocalization with Lamp-2 (Fig 5C and 5D) shows that bacterial recombinant ApoD reaches the LELC, but the signal disappears with a fast time course. Using immunoblot, we have estimated that only 30% of endocytosed native human ApoD is lost during a division cycle in HEK293T cells (Fig 5E and 5F), and a fraction of that loss corresponds to ApoD secretion to the culture medium (S3B Fig). These experiments demonstrate a very stable presence of native ApoD in the intracellular compartments analyzed above, and particularly in LELC, which suggests a functional role there. It is well known that proteins with essential functions within lysosomes bear carbohydrate shields against proteolysis [52]. In addition to the stable Lipocalin folding, ApoD N-linked glycosylation [53] might be responsible for such biochemical stability in protease-rich environments. To further assay whether the Lipocalin folding can be stable in the acidic lysosomal lumen, we performed ligand binding assays at pH 7. 0 and 5. 1 (Fig 5G; pH chosen in light of the pH distribution of ApoD-positive lysosomes under stress conditions, see below). Binding to retinoic acid, a generic hydrophobic ligand known to bind all human Lipocalins tested so far [19], indicates that ApoD ligand binding is functional under acidic conditions. An acidic pH is a defining functional property of lysosomes that allow their protease and lipase activities to be tightly controlled within the cell in addition to influence lysosomal fusions and traffic. We set to study the functional relationships of ApoD to lysosomal pH by using the membrane permeable LysoSensor Yellow/Blue DND-160, a ratiometric dye specifically targeted to all lysosomes, and not only those reaching the LELC through the endocytic pathway ([54,55]; see Methods and S5 Fig). We used either excitation analysis in cell populations (S5A Fig), suitable for pH 4. 0–6. 0, or emission spectral analysis of single lysosomes in confocal microscopy optical sections combined with ApoD immunodetection (S5B–S5D Fig; Linear fit for pH 4. 0–5. 5). We first analyzed the effects of the treatments used in our experimental paradigm on the average lysosomal pH in 1321N1 cell populations (Fig 6A). Chloroquine (CQ) was used as positive control. Two hours of PQ treatment resulted in significant lysosomal alkalinization (with an average increase of 0. 5 pH units), in agreement with the reported sensitivity of lysosomal membranes to oxidative stress [14] resulting in proton leakage that counteracts pH gradient generation mechanisms. When pH was measured at the single organelle level combined with ApoD labeling, we discover a striking difference in the frequency distributions of pH values (Fig 6B). Lysosomes without ApoD show a narrow pH distribution with a frequency maximum at pH 4. 4, while ApoD-positive lysosomes show a broaden distribution (range: 4. 4–5. 5) and a mode at pH 4. 7 still in the range of lysosomal pH. This difference observed in control conditions is maintained when lowering serum in the culture medium. When we apply PQ, ApoD-negative lysosomes suffer a mild alkalinization (average peak shifts 0. 2 pH units; Fig 6C) that is persistent after 24h of treatment. However, when the ApoD-positive lysosome pool is analyzed (Fig 6D), a larger pH increase is observed (average of 0. 5 pH units). This alkalinization is transitory, since lysosomal pH distribution returns after 24h of PQ treatment to more acidic values, within the range of ApoD-positive lysosomes in control conditions. These data support the existence of subsets of lysosomes differing not only in their pH and ApoD content, but also in their sensitivity and response to an oxidative insult. Our results show that ApoD is present in a subset of lysosomes specifically sensitive to oxidative stress that undergo a large but reversible alkalinization in response to PQ. Is ApoD responsible for these changes or it locates in lysosomes passively subjected to the PQ-triggered pH shifts? To test whether there is a causal relationship between ApoD presence and lysosomal pH we added exogenous purified human ApoD to non-expressing HEK293T cells, either simultaneous to PQ exposure (2h treatment) or sequentially, by first provoking lysosomal alkalinization with PQ (2h) and then adding exogenous ApoD (Fig 7A). We have already shown that by 2h ApoD has entered the LELC in HEK293T cells (Fig 5D). The addition of ApoD prevents the PQ-triggered alkalinization and is able to reverse an already established effect of PQ (asterisks Fig 7B). We also added purified ApoD to differentiated SH-SY5Y neurons (Fig 7C). Here a longer simultaneous ApoD-PQ treatment was applied due to the dynamics of ApoD entry into neuronal LELC (see below, Fig 8). Again, ApoD is able to prevent (particularly prominently in the long 24h treatment), as well as to revert, the alkalinizing effects of PQ (asterisks in Fig 7C). Lack of ApoD labelling in the absence of exogenous addition of hApoD indicates that SH-SY5Y neurons do not express ApoD, as has been recently confirmed by RNAseq techniques (http: //systemsbiology. uni. lu/shsy5y/). Therefore, the observed rescue of lysosomal pH can only be attributed to an endocytosis-mediated mechanism (Fig 8). Aside of modifying ApoD presence by exogenous addition, we cultured murine primary astrocytes from wild type (WT) or knock-out (ApoD-KO) mice (Fig 7D). Using this model, we demonstrate: 1) The PQ-dependent alkalinization and re-acidification of 1321N1 lysosomes is also present in WT primary astrocytes. 2) ApoD-KO lysosomes do increase their pH upon oxidative stress, therefore indicating that the alkalinization itself is not related to ApoD presence. 3) No re-acidification is achieved in the absence of ApoD. Summarizing, our data show that ApoD is present in a particular subset of PQ-sensitive lysosomes, which inevitably alkalinize in the presence of oxidative stress, an effect known to be due to lysosomal membrane damage [14,56]. We also show that ApoD is responsible for the pH recovery of PQ-challenged lysosomes. These data support the hypothesis that lysosomal membrane recovery is compromised in the absence of ApoD, and that this Lipocalin, with its structure and lipid-binding properties preserved, contributes to the repair of damaged lysosomal membranes in astrocytes and neurons. We have shown that ApoD affects the differential response of a subset of lysosomes to oxidative stress by actively promoting pH recovery. Then, is the entrance of ApoD to the lysosomal compartment a constitutive or a regulated process? A Lamp-2/ApoD colocalization analysis in differentiated SH-SY5Y neurons after a two-hour pulse of exogenous human ApoD (Fig 8A and 8B) shows that, in contrast with the observed rapid entrance into the LELC compartment in HEK293T cells (Fig 5), differentiated neurons show no colocalization in control conditions 2h after ApoD addition (Fig 8A and 8B). Colocalization is evident later on, after a 24h chase (Fig 8B). In contrast, a significant colocalization is observed at 2h in the presence of PQ, and keeps increasing during the 24h post-ApoD supplementation period (Fig 8B). Thus, ApoD entry into lysosomes in neurons is dependent on stress conditions, being slow in the absence of stress but very quick in its presence. A similar phenomenon is observed with native murine ApoD in primary astrocytes (Fig 8C), though with a remarkable feature: In control condition WT astrocytes show a clear predominant plasma membrane localization of ApoD and no colocalization with Lamp-2. ApoD entrance into the LELC is PQ-dependent and has a fast time course, with a substantial colocalization at 2h. We can conclude that the accelerated targeting of ApoD to the lysosomal compartment of neurons and primary astrocytes under stress conditions is a regulated process. To further explore the consequences of this process we measured the levels of 4-hydroxynonenal (4HNE) (Fig 9A, 9B and 9D), a lipid peroxidation-derived adduct, as a proxy for oxidative stress in each experimental condition. Interestingly, primary WT astrocytes have very low 4HNE levels in control conditions; they increase their oxidation levels by 2h of PQ treatment, but return to basal levels by 24h (Fig 9A and 9D). By contrast, ApoD-KO astrocytes show a significant basal level of oxidative stress and fail to recover from the insult (Fig 9B and 9D), further demonstrating the reported protective role of ApoD upon oxidative stress [26]. Starting with a higher oxidation state, the level achieved by 2h PQ in ApoD-KO astrocytes matches WT levels, indicating that cells have reached a 4HNE maximum. Therefore, ApoD entrance into lysosomes of WT astrocytes occurs at the peak of oxidative stress. If ApoD targeting to lysosomes is regulated by oxidative stress, we wondered why there is a significant colocalization of ApoD with Lamp2 in 1321N1 astrocytoma cells under control conditions (Figs 2–4). Fig 9C demonstrates that 1321N1 cells have a high basal oxidative stress, coherent with the high metabolic rate of cancer cells. Thus, ApoD is targeted to lysosomes under these conditions. The levels of 4HNE further increase in response to PQ treatment, and are followed by a substantial clearance of 4HNE adducts. This rebound effect is consistent with the pH recovery effect observed in our single-lysosome study (Fig 6D), and suggests that oxidative-challenged cells transiently activate protective mechanisms (ApoD among them) that clear lipid peroxidation products. An additional analysis of the 1321N1 single lysosome data (Fig 9E) shows a low proportion of ApoD-positive lysosomes in control conditions, and a significant early increase upon PQ treatment that is maintained by 24h (overriding the proportion of ApoD-negative lysosomes). Thus, the mechanism controlling lysosomal targeting of ApoD also occurs in the astrocytoma cell line 1321N1. The efficient clearance of oxidized products might be the result of a very efficient lysosomal function, contributed by the ApoD-dependent re-acidification after PQ insult. An active role of ApoD in oxidized lysosomes was further demonstrated by measuring lipofuscin, a reported readout of lysosomal pH dysfunction that results in cellular accumulation of damaged oxidized macromolecules [57]. A spectral analysis of confocal microscopy images of WT and ApoD-KO primary astrocytes (Fig 9F and 9G) shows that the absence of ApoD generates a significant increase in lipofuscin signal in astrocytes. If the mechanism by which ApoD controls lysosomal pH is due to lysosomal membrane stabilization, two predictions can be made for lysosomal behavior in the absence of ApoD: 1) lysosomal proteases would decrease their activity, and 2) cytoplasmic proteins would aberrantly enter lysosomes. A Cathepsin B activity assay (Fig 10A–10C) demonstrate the first prediction. In WT astrocytes (Fig 10A and 10C), PQ-triggered oxidation reduces Cathepsin B activity, but a clear recovery takes place upon prolonged PQ exposure in parallel with lipid-peroxide clearance (Fig 9A and 9C). In the absence of ApoD (Fig 10B and 10C), Cathepsin B activity is significantly reduced in basal conditions, further deteriorated upon PQ treatment, and no recovery is obtained after 24 h treatment. Galectin-3 immunocytochemistry (Fig 10C) demonstrate the second prediction. This lectin shows a diffuse cytoplasmic location in control WT astrocytes cultures, but translocates to leaky lysosomes undergoing oxidative stress-dependent membrane permeabilization upon PQ insult, giving a punctate labeling pattern [58]. Astrocytes lacking ApoD show evident Galectin-3 puncta in control conditions (arrows in Fig 9H), and the effect is further increased upon 2h PQ. In summary, entrance of ApoD into the LELC, and particularly into lysosomes, is actively promoted in pro-oxidative conditions in neurons and astrocytes. Our data demonstrate that ApoD, contributing to lysosomal pH recovery upon oxidative stress, is recruited to a vulnerable subset of lysosomes, where it helps to keep lipid peroxides levels under control and to safeguard lysosomal functional integrity by avoiding lysosomal membrane permeabilization. These findings represent a novel function for ApoD, and for Lipocalins. Their active role in the lysosomal compartment provides a clear explanation for the mechanism of GLaz rescue of polyglutamine-based neurodegeneration [37]: it requires fusion of autophagosomes to healthy lysosomes to optimize autophagy (Fig 1). Moreover, we have reported an protective role for ApoD in the functional recovery of injured mammalian peripheral nerves by a mechanism regulating myelin phagocytosis efficiency [32]. Taking into account our current results, the mechanistic link between these two apparently unrelated biological processes is clear: The direct control by ApoD of lysosomal function efficiency. We have centered this work on astrocytes, the front line of defense against oxidative stress and one of the nervous system cell types that express ApoD. Since ApoD conditions the pH-dependent functionality of the lysosomal compartment, how does it affect biological processes where a lysosomal optimal function is important for astrocytes? Recent studies reveal that astrocytes have phagocytic functions [59,60,61]. They digest the phagocytosed cargo through a process regulated by lysosome pH levels and autophagosome-lysosome fusion [62]. This is a slow process in astrocytes, and is proposed to regulate antigen presentation by these cells through lysosomal fusion to the plasma membrane. We have shown that lysosomal ApoD either enters autophagolysosomes or traffics back to the plasma membrane (Fig 3), and that lysosomal pH depends on the presence of ApoD (Figs 6 and 7). Therefore, ApoD might be a candidate regulator for the “digest-or-present” process in astrocytes. Astrocytes are reported to start degrading phagocytosed cargo in acidic lysosomes at 6h after exposure to cell debris [62]. We thus exposed primary astrocytes to DiI-labeled myelin for 3 days and monitored DiI signal at 2 and 6 days after myelin removal (Fig 11). We estimated the phagocytosis potential of WT and ApoD-KO astrocytes by measuring the number and size of DiI-labeled myelin particles (see Methods). Starting with comparable initial levels of phagocytic activity (Fig 11A; no differences are observed in the number of myelin particles phagocytosed during the 3h exposure period), both astrocyte genotypes decrease the number of particles over time. However, this reduction in numbers is accompanied by a significant increase in large myelin particles at 6 days post-myelin exposure in ApoD-KO astrocytes only (Fig 11B, 11E and 11F), indicating that phagocytosis resolution is impaired in the absence of ApoD. These results agree with the high load of phagocytosed material observed in alkaline astrocyte lysosomes [62]. Taking into account that myelin phagocytosis induces ROS production [63], the more alkaline ApoD-KO lysosomes are expected to be less efficient, resulting in delayed myelin degradation. No differences are found at earlier times, suggesting that the absence of ApoD results in a deregulated processing of the already ingested myelin, requiring the maintenance of lysosomal optimal pH and functionality.
This study demonstrates that ApoD contributes significantly to the evolutionarily conserved mechanism of protecting cells by protecting their lysosomes. ApoD could be the first lysosomal marker known to be specific for a particular subset of lysosomes: the most vulnerable to oxidative stress. Therefore, ApoD localization assessment should provide a useful tool for characterizing many physiological and pathological situations. Our work focuses on the endogenous mechanisms of protection in the nervous system, where astrocytes are central players and neurons are especially vulnerable cells. However, the known functional pleiotropy of ApoD warrants that it will be relevant to many other biological processes and pathological situations. Understanding ApoD actions in lysosomes will open the possibility of manipulating this mechanism for therapeutic purposes, using ApoD as a carrier to reach the lysosome. Although previously considered to play a lipid transport function in various body fluids, the Lipocalin ApoD can now be recognized as a relevant acute phase protein contributing to the nervous system response to stress, injury, neurodegeneration and aging by stabilizing the membrane of vulnerable lysosomes. This finding identifies a new lipid binding protein-dependent cellular mechanism by which lysosomes are functionally protected against oxidative stress.
The cell lines 1321N1, HEK293T, and SH-SY5Y were obtained from Sigma-Aldrich and ATCC. Cells were grown at 37°C in humidity-saturated atmosphere containing 5% CO2. The culture medium was replaced twice a week, and cells were subcultured at 90% confluence. The human astrocytoma cell line 1321N1 was cultured in Dulbecco-modified Eagle' s medium (DMEM; Lonza), supplemented with heat-inactivated 5% fetal bovine serum (FBS), 1% L-glutamine (final concentration 2 nM), and 1% P-S-A stock (final concentration: 100 U/ml penicillin, 100 U/ml streptomycin, 0. 25 μg/ml amphoterycin B). The human neuroblastoma cell line SH-SY5Y was cultured in DMEM supplemented with 4. 5 g/l glucose, heat-inactivated 10% FBS, 1% L-glutamine, 1% P-S stock (final concentration: 100 U/ml penicillin, 100 U/ml streptomycin) and 1% nonessential amino acids (Lonza). SH-SY5Y cells differentiation was achieved by culture on collagen-treated plates with medium supplemented with 3% FBS and Retinoic acid (10 μM). A 72h differentiation period was allowed before experiments. HEK293T cells were cultured in DMEM supplemented with 4. 5 g/l glucose, 1% L-glutamine, 1% P-S-A, and 10% FBS. For the exogenous addition of ApoD, human ApoD purified from breast cystic fluid [39] or recombinant human ApoD from E. coli (ProSpec) were added (10 nM) to the cell cultures for 2h. Cells treated with Paraquat (PQ; 500 μM; 1-24h), Chloroquine (CQ; 20 μM; 1h), Rapamycine (Rap; 2 μM; 2h), and 3-Methyladenine (3-MA; 5 mM; 2h) were cultured in phenol red-free DMEM supplemented with 1% L-glutamine, 1% P-S, and 0. 2% charcoal stripped FBS. This medium without additives was used as our low-serum (LS) condition. The expression constructs used in this work (pcDNA3. 1-ApoD; pHSVer-GA; pHSVGA-cox8; pCDNA3. 1-tgoGAm) were transiently transfected into cell lines using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s protocol. ApoD-KO mice were generated by homologous recombination [29] maintained in positive pressure-ventilated racks at 25±1°C with 12 h light/dark cycle, fed ad libitum with standard rodent pellet diet (Global Diet 2014; Harlan Inc. , Indianapolis, IN, USA), and allowed free access to filtered and UV-irradiated water. In order to avoid potential maternal effects of ApoD, and to generate WT and ApoD-KO mice of homogeneous genetic background, the experimental cohorts used in this study are the F1 generation of homozygous crosses of ApoD −/− and ApoD +/+ littermates born from heterozygous crosses of an ApoD-KO line backcrossed for over 20 generations into the C57BL/6J background. We used neonatal (0–1 days old) mice of two genotypes: ApoD-KO and their WT littermates. Cerebral cortices were quickly extracted, their meninges removed by rolling on a sterile filter paper, and pieces of cortex placed in Earle’s Balanced Salt Solution (EBSS) containing 2. 4 mg/ml DNAse I and 0. 2 mg/ml bovine serum albumin (BSA). Tissue was minced with a surgical blade; centrifuged (200 g, 2 min); incubated with 10 mg/ml trypsin for 15 min at 37°C (incubation terminated by 10% FBS addition); mechanically dissociated with a Pasteur pipette and centrifuged (200 g, 5 min). The last two steps were repeated, and the resulting cells were resuspended in DMEM with 10% FBS, 1% L-glutamine, 1% P-S-A. Cells were plated onto culture flasks and incubated at 37°C in 5% CO2 with 90–95% humidity, and the culture medium was replaced weekly. Cell cultures were used for experiments after two subculture steps, when >99% of cells are astrocytes [26]. Cells attached to poly-L-lysine (SIGMA) treated coverslips were fixed with 4% phosphate-buffered formaldehyde. Following washes in phosphate-buffered saline (PBS), the cells were blocked and permeabilized with Tween-20 (0. 1%) and 1% non-immune (goat or donkey) serum. Cells were incubated overnight at 4°C with the following primary antibodies. Rabbit serum anti-human ApoD [custom made by Abyntek Biopharma against purified ApoD [39], or generated by Dr. C. López-Otin]. Goat serum anti-mouse ApoD (Santa Cruz Biotechnology). Mouse serum anti-clathrin LCA (Santa Cruz Biotechnology). Mouse serum anti-caveolin-1 (Santa Cruz Biotechnology). Goat serum anti-catalase (Santa Cruz Biotechnology). Mouse serum anti-EEA1 (BD Biosciences). Mouse serum anti-LC3 (MBL). Rat monoclonal anti-Galectin-3 (American Type Culture Collection, ATCC). Goat serum anti-4HNE (Alpha Diagnostic). For immunolabeling with mouse monoclonal anti-human Lamp-2 and rat monoclonal anti-mouse Lamp-2 (DSHB), cells were blocked with 1% BSA, 10% normal goat serum, and 0. 1% saponin in PBS, and incubated with primary and secondary antibodies for 1 h at room temperature. Alexa Fluor 594 and 488 (Jackson Labs) or DyLight 405 (Thermo Scientific) -conjugated IgGs were used as secondary antibodies for fluorescence immunocytochemistry. After washes in PBS, the preparations were mounted with EverBrite Mounting Medium with DAPI, and sealed with CoverGrip Coverslip Sealant (Biotium). Cell lysates, or culture media (either directly or concentrated twenty times by 0. 22 μm filter centrifugation), were collected to analyze the amount of ApoD. Immunoblot analyses were performed with proteins transferred to PVDF membranes using standard procedures, and exposed to rabbit serum anti-human ApoD and HRP-conjugated goat-anti-rabbit (Santa Cruz Biotechnology). An HRP-conjugated anti-β actin antibody (Sigma) was used to normalize protein loads. Membranes were developed with ECL reagents (Millipore), and the signal visualized with a digital camera (BioRad). The integrated optical density of the immunoreactive protein bands was measured in images taken within the linear range of the camera, avoiding signal saturation. These methods were performed as previously described [39]. Fluorescence measurements were conducted with a Shimadzu RF-5301PC spectrofluorometer in a quartz cuvette (105. 251-QS, 3-mm path length; Hellma). Temperature was held at 22±0. 1°C. Excitation wavelength was 295 nm (selective for tryptophan residues). Emission was recorded at 327–400 nm with slit width set at 5 nm. Purified human ApoD was diluted to 0. 5 μM with 10 mM phosphate buffer (binding at pH 7. 0), or 30 mM sodium citrate (binding at pH 5. 1). The ligand retinoic acid (RA) was dissolved in dimethylformamide (DMF; Sigma). The mixture was equilibrated for 3 min in the dark before the fluorescence was recorded. The fluorescence spectrum in the presence of ligand was subtracted from DMF baseline obtained mixing the protein with the same amounts of carrier without ligand. Binding was assayed with RA 5 μM (1: 10 protein: ligand concentration) and a DMF final concentration of 0. 005%. Labeled cells were visualized with an Eclipse 90i fluorescence microscope (Nikon) equipped with a DS-Ri1 (Nikon) digital CCD camera. Images were acquired under the same conditions of illumination, diaphragm and condenser adjustments, exposure time, background correction and color levels. Confocal images were obtained with a 63x oil immersion objective (HCX PL Apo CS NA = 1. 4; Leica) attached to a confocal DMI 6000B microscope with a TCS SP5 confocal system (Leica) equipped with AOBS and AOTF systems. Fluorophores were excited with WLL laser (Leica) and a 405 line (Leica) controlled by LAS AF software (Leica). Emissions were collected with the AOBS system and three spectral detectors. Laser power and detection gains were set by scanning control samples labeled with secondary antibody alone. We ensured to obtain similar dynamic ranges in our images, and adjusted gain and offset using LUTs. In this manner, bleedthrough can be neglected. Negative control images showed very weak and homogeneous background. We obtained confocal sections under constant conditions to minimize image acquisition variation. Images were stored as 1024x1024 pixels and 8-bit TIFF files. Z-series (xyz scan) were performed. The number of z-stacks was determined by observing the limits of the cell membrane. The focus plane was set to be 3 μm beneath the section surface. The optimal value of the step size was calculated for the wavelength used to fulfill the Nyquist theorem. The optical section thickness was 0. 772 μm. Besides, images were taken with a 4x zoom, reducing field size. Pixel size corresponded to 0. 06*0. 06*0. 3777 μm3. Scanning was performed with a 1. 0 Airy unit pinhole size. Images were processed with a Gaussian Blur filter [Sigma (Radius): 1. 00], to facilitate object detection, and analyzed with the Colocalization Indices plug-in [50] and the 3D Object Counter tool using FIJI software. To analyze triple-colocalization experiments we used the Image Calculator and 3D Object Counter tools of FIJI. A principal component analysis (PCA) was performed on the 54 different variables per cell retrieved from our image analysis to reduce its dimensionality. Nine components were found with informative value, and three of them explained over 55% of the data variability. Intensity correlation quotient (ICQ) [49] variables were heavily represented in the first component. Pixel overlap proportions, and number and volume of objects were also of interest. Thus, we run a second PCA with 11 variables (S1A Fig). ICQ (variables 1–3) and Pixel Overlap (variable 4) presented the largest weight in component 1 (accounting for 31. 5% of variability), and relative Overlaps (variables 5–7) were the most important for component 2 (explaining 21. 3% of variability). The first component score of this PCA was used to assess for global statistical differences due to the experimental conditions (S1B Fig). Only EEA1 and Lamp2 showed significant variation between control and PQ condition (Two-way ANOVA, Holm-Sidak post-hoc method, p < 0. 05). This multivariate analysis helped us to focus on two main colocalization variables relative to ApoD signal (1 and 5; arrows in S1A Fig) and the number and volume of ApoD-positive objects (variables 8 and 11) to understand the dynamics of ApoD traffic in the cell. It also helps us to focus on the PQ-dependent changes, since most variables covariate between control and LS conditions in our two-way ANOVA study. Lysosomal pH was measured using the dye LysoSensor Yellow/Blue DND-160 (Life Technologies) as described [71,72,73,74]. As a ratiometric dye, the LysoSensor readout is independent of concentration. Moreover, because it is membrane permeable, its readout is representative of a broad range of lysosomes in comparison with a dextran-tagged probe that reach lysosomes by endocytosis. Experimental parameters such as incubation time and dye concentration have been set to minimize variation and to give the best signal-to-noise ratio [74]. Cells were grown to >80% confluence in black 96-well plates (NUNC), plated in alternating rows to control for any signal variation across the plate. After removal of medium and washes with PBS, cells were incubated for 3 min with 2 μM LysoSensor Yellow/Blue in isotonic solution (NaCl, 105 mM; KCl, 5 mM; HEPES-Acid, 6 mM; Na-HEPES, 4 mM; NaHCO3,5 mM; mannitol, 60 mM; glucose, 5 mM; MgCl2,0. 5 mM; CaCl2,1. 3 mM; pH adjusted to 7. 4). Dye loading and incubation steps were carried out at room temperature in the dark. After 3 min, cells were rinsed three times in isotonic solution, and incubated with either additional isotonic solution or with pH calibration buffers. After 10 min fluorescence was measured with a GENios Pro Fluorometer and recorded using the XFluor4GENiosPro software package (TECAN). Lysosomal pH was determined from the ratio of excitation light at 340 nm and 390 nm (F340 nm/F390 nm, 535 nm emission) [16]. Mean light levels at both excitation wavelengths were integrated over 2000 μs and recorded for each well. This step was repeated after 5 ms for each sample. The final calculated pH represents the mean of six measurements taken strictly 12 min after dye removal, as LysoSensor generates lysosomal alkalinization with longer incubation times. Absolute pH levels were obtained by calibrating lysosomal pH against standards. Cells present in calibration wells were incubated with 15 μM monensin and 30 μM nigericin (SIGMA), proton-cationophores that permeabilize the lysosomal membrane to Na+ and K+, respectively. These ionophores were added in a solution of 20 mM MES (2- (N-Morpholino) ethanesulfonic acid), 110 mM KCl and 20 mM NaCl, with pH 4. 0,4. 5,5. 0,5. 5 and 6. 0, forcing lysosomes to equilibrate with those pH values (S5A Fig). For LysoSensor staining, cells attached to poly-L-lysine coverslips were washed in warm isotonic solution, and incubated with isotonic solution or pH calibration buffers. Confocal fluorescence images were then obtained by exciting at 405 nm, and the emission collected at 420–700 nm (λ scan (xyz) ) taking two averaged images every 10 nm. Using the LAS AF Lite software (LEICA), we created ROIs for each lysosome and their emission spectra were obtained. Each spectrum was fitted to a five parameters Weibull’s equation (S5B and S5D Fig). The emission 470/524 nm ratio was then calculated for each sample. The lysosomal pH values were determined from the standard curve generated with the pH calibration samples (S5C Fig). Before cells were fixed and permeabilized a white field image was taken in the confocal microscope (S5D Fig step 4). Immunocytochemistry was then performed to detect ApoD in lysosomes, and each cell was position-identified to overlap ApoD to LysoSensor signals (S5D Fig steps 5–7). Images were analyzed with FIJI, and estimate the proportion of ApoD-positive and negative lysosomes we used the Image Calculator tool. Lysosomal Cathepsin B activity was measured using the Magic Red assay as directed by manufacturer (InmmunoChemistry Technology, LLC). Briefly, cells are exposed in vivo to a membrane-permeable non-fluorescent substrate that yields a red-fluorescent product upon enzymatic cleavage. Both, confocal microscopy (590 nm excitation / 630–640 nm emission) and cell population analysis in 96-well plates (570 nm excitation / 612 nm emission) were performed. L-leucyl-L-leucine methyl ester (Cayman Chemical), 2. 5 mM, was used as positive control for lysosomal membrane rupture. 1321N1 cells, under control or PQ conditions, destined for pre-embedding immunogold labeling of ApoD were fixed in 4% formaldehyde and 0. 3% glutaraldehyde in 0. 1 M phosphate buffer (PB) pH 7. 4 for 30 min at 4°C. Following washes in 0. 1 M PB, the cells were blocked with 0. 1% cold water fish skin gelatin and permeabilized with Tween-20 (0. 5%) in Tris-buffered saline (TBS; 20 mM Tris-HCL, 150 mM NaCl). Cells were incubated for 48h at 4°C with rabbit serum anti-human ApoD primary antibody [custom made by Abyntek Biopharma against purified ApoD [39]] diluted 1: 500 in TBS containing Tween-20 (0. 5%) and 0. 1% cold water fish skin gelatin. Cells were later washed several times and incubated with ultra-small gold-conjugated goat anti-rabbit secondary antibodies (EMS, Electron Microscopy Sciences) diluted 1: 50 in PBS for 48h at 4°C. After several washes with PBS, cells were post-fixed in 2% glutaraldehyde in PBS for 20 min, washed and the ultra-small gold particles were silver enhanced for 20 min at room temperature with AURION R-Gent SE-EM (Silver Enhancement for Electron Microscopy) (EMS, Electron Microscopy Sciences) following manufacturer’s indications. Later, cells were post-fixed with 0. 5% OsO4 in PBS for 20 min at 4°C and washed with PBS. Cells were then dehydrated through a graded series of ethanol, and embedded in Epoxy EMbed-812 resin (EMS, Electron Microscopy Sciences). Ultrathin sections were obtained with an Ultracut E ultramicrotome (Reichert/Leica), contrasted with uranyl acetate and lead citrate, and analyzed using a JEOL JEM-1011 HR electron microscope with a CCD Gatan ES1000W camera with iTEM software. Myelin isolation and labeling were previously described [32]. Fluorescence microscopy was used to visualize myelin uptake by astrocytes. For this purpose, 2. 5×105 cells were cultured on 12 mm coverslips at 37°C in 5% CO2. Cells were incubated with 5 μg of DiI-labeled myelin (10 μg/ml) for three days. After incubation, non-endocytosed and unbound myelin was removed by washing twice with PBS, and cells were grown at 37°C for 2 or 6 days. Cells were fixed in 4% formaldehyde in PBS, washed in PBS and mounted with EverBrite Mounting Medium with DAPI. DiI-labeled particles were visualized with a fluorescence microscope as described above. Number and size of myelin particles ingested by cultured astrocytes were measured from thresholded images with FIJI. Flies were grown in a temperature-controlled incubator at 25°C, 60% relative humidity, under a 12 h light—dark cycle. They were fed on wet yeast 84 g/l, NaCl 3. 3 g/l, agar 10 g/l, wheat flour 42 g/l, apple juice 167 ml/l, and propionic acid 5 ml/l. Fly females were used in all experiments. We used the line gmr: GAL4 to drive transgenes expression to the eye photoreceptors. UAS: hATXN182Q was used to trigger the neurodegenerative phenotype [75], combined with UAS: GLaz [37] and/or UAS: Dor-RNAi [48]. Recombination of two of the elements present in the second chromosome (gmr: GAL4 and UAS: GLaz) was required to obtain some of the experimental combinations. Two independent lines were used. Flies were anesthetized with CO2 and immobilized with adhesive tape. Fly eyes were photographed with a Nikon DS-L1 digital camera, in a Nikon SMZ1000 stereomicroscope equipped with a Plan Apo 1× WD70 objective. Local intensity maxima were obtained with the FIJI program, and nearest neighbor distances were calculated for each ommatidium. A regularity index (IREG) was estimated as described [47], and a percent recovery was calculated considering 0% the average degenerated eye and 100% the control wild type eye. Samples of 20–35 flies (3 days old) per condition and genotype were used to calculate the mean±SEM regularity index. We have used the freely available FIJI plug-in FLEYE for automated analysis of fly eye pictures [47]. Experimental procedures applied to mice were approved by the University of Valladolid Animal Care and Use Committee, and followed the regulations of the Care and the Use of Mammals in Research (European Commission Directive 86/609/CEE, Spanish Royal Decree ECC/566/2015). Statistical analyses were performed with SPSS v. 19 (IBM) and SigmaPlot v. 11. 0 (Systat) softwares. A p value < 0. 05 was used as a threshold for significant changes. The tests used for each experiment are stated in figure legends.
This work is the result of our search for the mechanism of action of Apolipoprotein D (ApoD), a neuroprotective lipid-binding protein that confers cell resistance to oxidative stress. ApoD is one of the few genes consistently over-expressed in the aging brain of all vertebrate species, and no nervous system disease has been found concurring without ApoD over-expression. All evidence supports ApoD as an endogenous mechanism of protection. We demonstrate here that this extracellular lipid binding protein is endocytosed and targeted in a finely controlled way to subsets of lysosomes in need of protection, those most sensitive to oxidative stress. ApoD reveals the existence of biologically relevant lysosomal heterogeneity that conditions the oxidation state of cells, their phagocytic or autophagic capacity, and the final output in neurodegenerative conditions. The stable presence of ApoD in lysosomes is sufficient and necessary for lysosomes to recover from oxidation-induced membrane permeabilization and loss of proton gradients. ApoD-mediated control of lysosomal membrane integrity represents a new cell-protection mechanism at the hub of many cellular functions, and is critical for the outcome of a wide variety of neurodegenerative diseases. Therapeutic opportunities open, by providing a route of entry and a repair mechanism for lysosomes in pathological situations.
cell death
lysosomes
autophagic cell death
oxidative stress
astrocytes
cell processes
neuronal differentiation
neuroscience
macroglial cells
cell differentiation
developmental biology
cellular structures and organelles
animal cells
glial cells
cell membranes
cellular neuroscience
cell biology
neurons
biology and life sciences
cellular types
Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress