Lysosomal Response Research Articles

Lysosomal TFEB-TRPML1 Axis in Astrocytes Modulates Depressive-like Behaviors.

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress-related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome-related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte-specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive-like behaviors by inhibiting lysosomal exocytosis-mediated adenosine 5'-triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive-like behaviors induced by astrocyte-specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress-sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants.

Knockout of the neonatal Fc receptor alters immune complex trafficking and lysosomal function in cultured podocytes.

Podocytes are key to preventing the filtration of serum proteins into the urine. Recent evidence also suggests that in immune mediated kidney diseases, podocytes are the targets of immune complexes (ICs). The mechanisms whereby podocytes handle and respond to ICs remain unknown. The neonatal Fc receptor (FcRn) is involved in IgG handling in podocytes and is also required in dendritic cells to traffic ICs to the lysosome for proteolytic degradation of antigen and presentation on MHC II. Here we examine the role of FcRn in handling ICs in podocytes. We show that knockout of FcRn in podocytes results in decreased trafficking of ICs to the lysosome and increases IC trafficking to recycling endosomes. FcRn KO also alters lysosomal distribution, decreases lysosomal surface area and decreases cathepsin B expression and activity. We demonstrate that signaling pathways in cultured podocytes differ after treatment with IgG alone versus ICs and that podocyte proliferation in both WT and KO podocytes is suppressed by IC treatment. Our findings suggest that podocytes respond differentially to IgG versus ICs and that FcRn modifies the lysosomal response to ICs. Elucidating the mechanisms underlying podocyte handling of ICs may provide novel pathways to modulate immune mediated kidney disease progression.

Aggregation-Induced emission photosensitizer with lysosomal response for photodynamic therapy against cancer

Photosensitizers play a key role in bioimaging and photodynamic therapy (PDT) of cancer. However, conventional photosensitizers usually do not achieve the desired efficacy in PDT due to their poor photostability, targeting ability, and responsiveness. Herein, we designed a series of photosensitizers with aggregation-induced emission (AIE) effect using benzothiazole- triphenylamine (BZT-triphenylamine) as the parent nucleus. The synthesized compound SIN ((E)-2-(4-(diphenylamino)styryl)-3-(4-iodobutyl)benzo[d]thiazol-3-ium) exhibits good biocompatibility, photostability, and bright emission in the near-infrared range (600–800 nm). The fluorescence emission intensity is responsive to viscosity, with significant fluorescence enhancement (48 times) and high fluorescence quantum yield (4.45 %) at high viscosity. Moreover, SIN has particular lysosome targeting properties with a Pearson correlation coefficient (PCC) of 0.97 and has good 1O2 generation ability under white light irradiation, especially in a weak acidic environment. Thus, SIN can realize good bioimaging ability and photodynamic therapeutic efficacy under the highly viscous and weakly acidic environment of lysosomes in the tumor cells. This study indicates that SIN has potential as a multifunctional organic photosensitizer for bioimaging and PDT of tumor.

Aggregation-Induced Emission Photosensitizer with Lysosomal Response for Photodynamic Therapy Against Cancer

Aggregation-Induced Emission Photosensitizer with Lysosomal Response for Photodynamic Therapy Against Cancer

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Neuron‐selective induction of granulovacuolar degeneration bodies: A lysosomal stress response to tau aggregation?

AbstractBackgroundGranulovacuolar degeneration bodies (GVBs) are membrane‐bound vacuolar structures harboring a dense core that accumulate in the brains of patients with neurodegenerative disorders, including Alzheimer’s disease and other tauopathies. Insight into the origin of GVBs and their connection to tau pathology has been limited by the lack of suitable experimental models for GVB formation.MethodWe used confocal, automated, super‐resolution and electron microscopy in different models for seeded tau aggregation.ResultWe demonstrate that the seeding of tau pathology triggers the formation of GVBs in mouse models in vivo and in primary mouse neurons in vitro. Seed‐induced intracellular tau aggregation, but not seed exposure alone, causes GVB formation in cultured neurons but not astrocytes. The extent of tau pathology strongly correlates with the GVB load. The in vitro tau‐induced GVBs are immunoreactive for the established GVB markers CK1δ, CK1ɛ, CHMP2B, pPERK, peIF2α and pIRE1α. GVBs contain a LAMP1‐ and LIMP2‐positive single membrane that surrounds the dense core and vacuole. The proteolysis reporter DQ‐BSA is detected in the majority of GVBs, demonstrating that GVBs contain degraded endocytic cargo. GFP‐tagged CK1δ accumulates in the GVB core, whereas GFP‐tagged tau or GFP alone do not, indicating selective targeting of cytosolic cargo to GVBs. In addition, GVB positive neurons have increased levels of stress related proteins.ConclusionWe established the first in vitro model for GVB formation by seeding tau pathology in primary neurons. The tau‐induced GVBs have the marker signature and morphological characteristics of GVBs in the human brain. We show that GVBs are lysosomal structures that are distinguished by the accumulation of a characteristic subset of proteins in a dense core. Our data indicate that GVB containing neurons are in a state of enhanced cellular stress.

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

Implications of Altered Endosome and Lysosome Biology in Space Environments.

Space exploration poses multiple challenges for mankind, not only on a technical level but also to the entire physiology of the space traveller. The human system must adapt to several environmental stressors, microgravity being one of them. Lysosomes are ubiquitous to every cell and essential for their homeostasis, playing significant roles in the regulation of autophagy, immunity, and adaptation of the organism to changes in their environment, to name a few. Dysfunction of the lysosomal system leads to age-related diseases, for example bone loss, reduced immune response or cancer. As these conditions have been shown to be accelerated following exposure to microgravity, this review elucidates the lysosomal response to real and simulated microgravity. Microgravity activates the endo-lysosomal system, with resulting impacts on bone loss, muscle atrophy and stem cell differentiation. The investigation of lysosomal adaptation to microgravity can be beneficial in the search for new biomarkers or therapeutic approaches to several disease pathologies on earth as well as the potential to mitigate pathophysiology during spaceflight.

A multibiomarker approach to assess lead toxicity on the black clam, Villorita cyprinoides (Gray, 1825), from Cochin estuarine system (CES), southwest coast, India.

The southwest coast of India along the Cochin region is seriously affected by metal contamination from increasing industrial activities. This contribution pertains to the chronic toxicity effects of lead and its biomarker responses on the black clam, Villorita cyprinoides, largely endemic to Southern India. The metal concentration (lead (Pb), copper (Cu), zinc (Zn), cadmium (Cd), nickel (Ni), and chromium (Cr)) and contamination indices such as geoaccumulation index and contamination factor suggests that the Cochin estuarine system (CES) is moderately to heavily polluted with metals. The 96h effective median lethal concentration (LC50) for Pb was 12.08mgL-1 at 95% confidence level, whereas the NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) were 0.46mgL-1 and 0.83mgL-1 respectively. The chronic toxicity value for Pb was 0.64mgL-1. Accumulation of Pb in V. cyprinoides after chronic toxicity test was several-fold higher than exposure concentration. On exposure to sublethal concentrations of lead, gills showed prominent pathological lesions such as damaged lateral cilia, congested hemolymph sinus, damaged abfrontal cilia, damaged ciliary filaments, fusion of gill lamellae, and reduced inter lamellar space. Filtration rate of clams exposed to Pb (86.69 ± 1.98ml clam-1h-1) was lower than that in control experiments (191.86 ± 6.48ml clam-1h-1). Antioxidant enzymes such as esterase (EST), superoxide dismutase (SOD), and malate dehydrogenase (MDH) exhibited different patterns in isoenzyme activity. Neutral red retention time (NRRT) showed a decrease from control to higher concentrations indicating membrane stability of hemocytic lysosomes decreased with the increasing metal exposure concentration. At LOEC and higher concentrations lysosomes showed enlargement and fragmentation. Lysosomal responses in V. cyprinoides can be used as a key cellular stress biomarker in assessing lead and other metal contamination.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis.

Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

Bioaccumulation, tissue and cell distribution, biomarkers and toxicopathic effects of CdS quantum dots in mussels, Mytilus galloprovincialis

The bioaccumulation, cell, tissue distribution, and biological effects of 5 nm glutathione-capped CdS quantum dots (CdS QDs) in mussels was compared to bulk and aqueous Cd forms through a two-tier experimental approach. In the 1st tier, mussels were exposed for 3 d to 0.05, 0.5 and 5 mg Cd/l (QDs, bulk, aqueous), bioaccumulation, distribution and lysosomal responses were investigated. In the 2nd tier, mussels were exposed for 21 d to the same forms at the lowest effective concentration selected after Tier 1 (0.05 mg Cd/l), biomarkers and toxicopathic effects were investigated. Accumulation was comparable in QDs and aqueous Cd exposed mussels after 3 d. After 21 d, QDs exposed mussels accumulated less than mussels exposed to aqueous Cd and localised in the endo-lysosomal system and released to the alveoli lumen (21 d) after exposure to QDs and aqueous Cd. Intracellular levels of Cd increased on exposure to QDs and aqueous Cd, and to a lesser extent to bulk, and accompanied by the up-regulation of metallothionein 10 (1 d) and 20 (1, 21 d). Lysosomal membrane destabilisation depended on Cd2+ released by all forms but was marked after exposure to aqueous Cd (1 d). Toxicopathic effects (vacuolisation, loss of digestive cells and haemocytic infiltration) were evident after exposure to QDs (1 d) and aqueous Cd (21 d). Toxicity most likely depended on the ionic load resulting from Cd2+ release from the different forms of Cd; yet nanoparticle-specific effects of QDs cannot be disregarded.

LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson's disease and Crohn's disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

WDR41 supports lysosomal response to changes in amino acid availability.

C9orf72 mutations are a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. The C9orf72 protein undergoes regulated recruitment to lysosomes and has been broadly implicated in control of lysosome homeostasis. However, although evidence strongly supports an important function for C9orf72 at lysosomes, little is known about the lysosome recruitment mechanism. In this study, we identify an essential role for WDR41, a prominent C9orf72 interacting protein, in C9orf72 lysosome recruitment. Analysis of human WDR41 knockout cells revealed that WDR41 is required for localization of the protein complex containing C9orf72 and SMCR8 to lysosomes. Such lysosome localization increases in response to amino acid starvation but is not dependent on either mTORC1 inhibition or autophagy induction. Furthermore, WDR41 itself exhibits a parallel pattern of regulated association with lysosomes. This WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling as constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation. Collectively, this study reveals an essential role for WDR41 in supporting the regulated binding of C9orf72 to lysosomes and solidifies the requirement for a larger C9orf72 containing protein complex in coordinating lysosomal responses to changes in amino acid availability.

Styrene impairs normal embryo development in the Mediterranean mussel (Mytilus galloprovincialis)

This study analysed the effects of styrene, a main monomer in plastic manufacturing and acknowledged to be amongst the most common plastic leachates, on early embryo development of the Mediterranean mussel. Embryotoxicity tests showed that styrene impaired normal embryo development at concentrations (0.01 μg/L–1 mg/L) encompassing the environmental range. Occurrence of normal D-veligers was significantly reduced up to 40% of the total, and larval size was reduced of about 20%. D-veligers grown in the presence of styrene (0.1 and 10 μg/L) showed significant reduction of total Multixenobiotic resistance (MXR) efflux activity that was not apparently related to transcriptional expression of genes encoding P-glycoprotein (ABCB) and Mrp (ABCC), the two main ABC transporters of embryonal MXR system. Indeed, ABCB transcription was not affected by styrene, while ABCC was up-regulated. At these same concentrations, transcriptional profiles of 15 genes underlying key biological functions in embryo development and potential targets of adverse effects of styrene were analysed. Main transcriptional effects were observed for genes involved in shell biogenesis and lysosomal responses (down-regulation), and in neuroendocrine signaling and immune responses (up-regulation). On the whole, results indicate that styrene may affect mussel early development through dysregulation of gene transcription and suggest the possible conservation of styrene mode of action across bivalve life cycle and between bivalves and humans, as well as through unpredicted impacts on protective systems and on shell biogenesis.

ZnT2 is critical for lysosome acidification and biogenesis during mammary gland involution.

Mammary gland involution, a tightly regulated process of tissue remodeling by which a lactating mammary gland reverts to the prepregnant state, is characterized by the most profound example of regulated epithelial cell death in normal tissue. Defects in the execution of involution are associated with lactation failure and breast cancer. Initiation of mammary gland involution requires upregulation of lysosome biogenesis and acidification to activate lysosome-mediated cell death; however, specific mediators of this initial phase of involution are not well described. Zinc transporter 2 [ZnT2 ( SLC30A2)] has been implicated in lysosome biogenesis and lysosome-mediated cell death during involution; however, the direct role of ZnT2 in this process has not been elucidated. Here we showed that ZnT2-null mice had impaired alveolar regression and reduced activation of the involution marker phosphorylated Stat3, indicating insufficient initiation of mammary gland remodeling during involution. Moreover, we found that the loss of ZnT2 inhibited assembly of the proton transporter vacuolar ATPase on lysosomes, thereby decreasing lysosome abundance and size. Studies in cultured mammary epithelial cells revealed that while the involution signal TNFα promoted lysosome biogenesis and acidification, attenuation of ZnT2 impaired the lysosome response to this involution signal, which was not a consequence of cytoplasmic Zn accumulation. Our findings establish ZnT2 as a novel regulator of vacuolar ATPase assembly, driving lysosome biogenesis, acidification, and tissue remodeling during the initiation of mammary gland involution.

How does temperature affect functional kleptoplasty? Comparing populations of the solar-powered sister-species Elysia timida Risso, 1818 and Elysia cornigera Nuttall, 1989 (Gastropoda: Sacoglossa)

BackgroundDespite widespread interest in solar-powered sea slugs (Sacoglossa: Gastropoda), relatively little is know about how they actually perform functional kleptoplasty. Sister-taxa Elysia timida and E. cornigera provide an ideal model system for investigating this phenomenon, since they feed on the same algal genus and only E. timida is capable of long-term kleptoplasty. Recent research has explored factors regarding functional kleptoplasty in E. timida, including their starvation longevity, digestive activity, autophagal response and photosynthetic efficiency under two different temperature conditions (18 °C and 21 °C). These studies revealed the trends E. timida displays regarding each factor during starvation as well as influences temperature has on some aspects of functional kleptoplasty. This study examines E. cornigera regarding each of these factors in an attempt to elucidate differences between each species that could explain their differing kleptoplastic abilities. Since both species naturally occur in 25 °C seawater (E. timida peak summer temperature, E. cornigera low winter temperature), each species was acclimatized to 25 °C to facilitate comparison and determine if these species exhibit physiological differences to starvation when under the same environmental conditions.ResultsWhen comparing the different E. timida temperature treatments, it becomes clear that increased temperatures compromise E. timida’s kleptoplastic abilities. Specimens acclimatized to 25 °C revealed shorter starvation longevities surviving an average 42.4 days compared to the 95.9 day average observed in specimens exposed to 18 °C. Each temperature treatment displayed a significantly different decrease throughout the starvation period in both, the rate of photosynthetic efficiency and in the decreasing functional kleptoplast abundance. Lysosomal abundances are assessed here as indicators of different aspects of metabolic activity, which could be correlated to temperature. E. cornigera, also acclimatized to 25 °C did not display significantly similar patterns as any of the E. timida temperature treatments, having fewer incorporated kleptoplasts, a higher lysosomal response to starvation, a faster decrease in photosynthetic efficiency and a lower starvation longevity.ConclusionsThese results confirm that each species has different physiological reactions to starvation and kleptoplast retention, even under the same conditions. While temperature affects aspects of functional kleptoplasty, it is likely not responsible for the differences in kleptoplastic abilities seen in these species.

Functional Involvement of Carbonic Anhydrase in the Lysosomal Response to Cadmium Exposure in Mytilus galloprovincialis Digestive Gland.

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme, whose functions in animals span from respiration to pH homeostasis, electrolyte transport, calcification, and biosynthetic reactions. CA is sensitive to trace metals in a number of species. In mussels, a previous study demonstrated CA activity and protein expression to be enhanced in digestive gland by cadmium exposure. The aim of the present work was to investigate the functional meaning, if any, of this response. To this end the study addressed the possible involvement of CA in the lysosomal system response of digestive gland cells to metal exposure. The in vivo exposure to acetazolamide, specific CA inhibitor, significantly inhibited the acidification of the lysosomal compartment in the digestive gland cells charged with the acidotropic probe LysoSensor Green D-189, demonstrating in vivo the physiological contribution of CA to the acidification of the lysosomes. Under CdCl2 exposure, CA activity significantly increased in parallel to the increase of the fluorescence of LysoSensor Green charged cells, which is in turn indicative of proliferation and/or increase in size of lysosomes. Acetazolamide exposure was able to completely inhibit the cadmium induced Lysosensor fluorescence increase in digestive gland cells. In conclusion, our results demonstrated the functional role of CA in the lysosomal acidification of Mytilus galloprovincialis digestive gland and its involvement in the lysosomal activation following cadmium exposure. CA induction could physiologically respond to a prolonged increased requirement of H+ for supporting lysosomal acidification during lysosomal activation.

Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy

Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinson's disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein in MSA.

Aβ42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer’s disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5–1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways.