Figure 4 - uploaded by Colin Anderson
Content may be subject to copyright.
Lysosomal dysfunction is not responsible for the observed p62 accumulation. (A) Representative blot of LC3-I, LC3-II, p62 and β-actin of a CTL and DS fibroblast cell line at basal levels, after CQ treatment, after SS and CQ co-treatment and after SS and h2o cotreatment used as control. (B) Quantification of fold change in abundance of LC3-II of a CTL and DS fibroblast cell line after CQ, SS+CQ and SS+h2o. (C) Quantification of fold change in abundance of p62 of a CTL and DS fibroblast cell line after CQ, SS+CQ and SS+h2o. (D) IF for LAMP2 (Green), p62 (Red) and DAPI (Blue) in a CTL and DS fibroblast cell line at basal levels or after SS. (E) Quantification of LAMP2/p62 co-localization (yellow) fluorescence intensity (area-pixels) after SS (% based on abundance of CTL SS). (F) Lysotracker-Red fluorescence intensity in a CTL and DS fibroblast cell line after SS (% based on fluorescence intensity of CTL SS). (G) Proteolytic activity of lysosomal cathepsin B (% based on activity of CTL SS), White arrowhead-p62 only puncta; Black arrowhead-p62/LAMP2 co-localized puncta.
Source publication
Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21 (Tri21). This unbalanced karyotype has the ability to produce proteotoxic stress and dysfunction of the proteostasis network (PN), which are mechanistically associated with several comorbidities found in the DS phenotype. Autophagy is the cellular process responsible for bu...
Contexts in source publication
Context 1
... if lysosomal dysfunction is responsible for the observed impairment of autophagic degradation in DS cells, SS co-treatment with CQ or SS alone should display similar levels of p62 and LC3-II in these DS cells. Although not statistically significant, we observed that CQ and SS co-treatment resulted in apparent increased abundance of p62 and LC3-II compared to SS treatment alone ( Figure 4A-C). The observation that p62 and LC3-II protein levels were decreased in the absence of CQ indicates that the lysosome is unlikely to be the source of the observed impairment of autophagic degradation. ...
Context 2
... is ubiquitously expressed in the lysosomes, it is a well characterized marker of lysosomal localization and is thought to promote membrane integrity for lysosomal stability (67). Our experiments showed that SS lead to partial co-localization of p62 with LAMP2A in DS cells ( Figure 4D-E), further supporting the AL accumulation observed in our TEM experiment. Presence of "p62-only" puncta and only partial p62 and LAMP2 co-localization . ...
Context 3
... fluorescence intensity was measured in CTL and DS fibroblast cell lines after SS, to investigate possible lysosomal pH disturbance. No significant difference in intensity was observed in the CTL or DS fibroblasts, indicating normal pH levels between the two groups ( Figure 4F). Additionally, the lysosome contains proteolytic enzymes called cathepsins (63,64). ...
Context 4
... our experiment, we evaluated cathepsin B function as an index of lysosomal degradation capacity since inhibition of activity may explain dysfunctional clearance in DS fibroblasts. Remarkably, cathepsin B activity did not differ significantly between CTL and DS cells after SS, indicating that the lysosome is not overtly dysfunctional in DS ( Figure 4G). TFEB is a transcription factor responsible for regulating lysosomal biogenesis (69). ...
Context 5
... further examined lysosomal dysfunction via assessment of activation of TFEB after SS by using a combination of IF and TFEB DNA-binding activity ELISA. Similar results regarding nuclear localization and DNA binding of TFEB were observed in both CTL and DS fibroblasts after SS (Supplemental Figure 4), again indicating that the defects in autophagic degradation in DS models after SS may not be explained by simple disruption of lysosomal physiology and signaling. Previous research has indicated that the autophagic process is subject to significant regulatory interaction with the endosomal trafficking system, which is heavily regulated by members of the Rab protein family(70). ...
Context 6
... if lysosomal dysfunction is responsible for the observed impairment of autophagic degradation in DS cells, SS co-treatment with CQ or SS alone should display similar levels of p62 and LC3-II in these DS cells. Although not statistically significant, we observed that CQ and SS co-treatment resulted in apparent increased abundance of p62 and LC3-II compared to SS treatment alone ( Figure 4A-C). The observation that p62 and LC3-II protein levels were decreased in the absence of CQ indicates that the lysosome is unlikely to be the source of the observed impairment of autophagic degradation. ...
Context 7
... is ubiquitously expressed in the lysosomes, it is a well characterized marker of lysosomal localization and is thought to promote membrane integrity for lysosomal stability (67). Our experiments showed that SS lead to partial co-localization of p62 with LAMP2A in DS cells ( Figure 4D-E), further supporting the AL accumulation observed in our TEM experiment. Presence of "p62-only" puncta and only partial p62 and LAMP2 co-localization . ...
Context 8
... fluorescence intensity was measured in CTL and DS fibroblast cell lines after SS, to investigate possible lysosomal pH disturbance. No significant difference in intensity was observed in the CTL or DS fibroblasts, indicating normal pH levels between the two groups ( Figure 4F). Additionally, the lysosome contains proteolytic enzymes called cathepsins (63,64). ...
Context 9
... our experiment, we evaluated cathepsin B function as an index of lysosomal degradation capacity since inhibition of activity may explain dysfunctional clearance in DS fibroblasts. Remarkably, cathepsin B activity did not differ significantly between CTL and DS cells after SS, indicating that the lysosome is not overtly dysfunctional in DS ( Figure 4G). TFEB is a transcription factor responsible for regulating lysosomal biogenesis (69). ...
Context 10
... further examined lysosomal dysfunction via assessment of activation of TFEB after SS by using a combination of IF and TFEB DNA-binding activity ELISA. Similar results regarding nuclear localization and DNA binding of TFEB were observed in both CTL and DS fibroblasts after SS (Supplemental Figure 4), again indicating that the defects in autophagic degradation in DS models after SS may not be explained by simple disruption of lysosomal physiology and signaling. Previous research has indicated that the autophagic process is subject to significant regulatory interaction with the endosomal trafficking system, which is heavily regulated by members of the Rab protein family(70). ...
Similar publications
Disturbed proteostasis as reflected by a massive accumulation of misfolded protein aggregates is a central feature in Alzheimer’s disease. Proteostatic disturbances may be caused by a shift in protein production and clearance. Whereas rare genetic causes of the disease affect the production side, sporadic cases appear to be directed by dysfunction...
Citations
... Consistently, we show that the NAD + depletion along with the aberrant ROS generation coincides with a suppressed expression of the Atg components, ATG7 and LC3 of the Atg12 and Atg8 systems and with a down-regulation of the phosphorylation of Beclin-1 for autophagic flux induction in DS fibroblasts (Fig. 13). Diminished autophagic clearance along with lysosomal impairments have been already reported in DS [65][66][67][68]. However, the novel aspect of the current study is that CBS inhibition normalizes some of these alterations, thereby further promoting the availability of the ATG3 component, possibly to facilitate the formation of functional complexes of the autophagic machinery (Fig. 13). ...
Background
Overexpression of the transsulfuration enzyme cystathionine-β-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters.
Methods
8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS.
Results
DS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. ¹³C glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with a dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters.
Conclusions
Increased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.
Intracellular protein trafficking via the endosomes plays a key role in the maintenance of normal neuronal function. Although many diseases of the central nervous system exhibit specific pathological hallmarks, abnormalities of the endosome system are common traits for several of them including Alzheimer's disease (AD). Three main routes originate from the endosomes: the recycling, the degradation and the retrograde pathway. Studies have shown that the majority of Down syndrome subjects develop AD pathology and manifest altered morphology and number of endosomes, abnormalities in lysosome acidification and exosome secretion suggesting that dysfunction of one of these pathways could play a functional role in the AD‐like phenotype of the syndrome. Two of the major endosomal routes are mediated by the retromer complex, a multimeric system responsible for transport of cargo from the endosome to the trans‐Golgi network or to the cell membrane. Recently, a new endosome system structurally related to the retromer, called “retriever’, has been reported. While we know a lot about the neuropathophysiology of the retromer complex, no precise pathogenic role for the retriever has been identified yet. Here, we will review the neurobiology of the endosome system and its role as key player in the development of AD‐like pathology in Down syndrome. Additionally, we will discuss current knowledge on these two main endosome systems, retromer and retriever, and their potential as novel therapeutic targets.
This article is protected by copyright. All rights reserved.
