Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis

α-synuclein dysregulation is a critical aspect of Parkinson''s disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson''s disease, thus connecting these aspects of Parkinson''s disease to the propagation of α-synuclein pathology in cells.     

Real‐Time Investigation of Intercalation and Structure Evolution in Printed Polymer:Fullerene Bulk Heterojunction Thin Films

The complex intermixing morphology is critical for the performance of the nanostructured polymer:fullerene bulk heterojunction (BHJ) solar cells. Here, time resolved in situ grazing incidence X‐ray diffraction and grazing incidence small angle X‐ray scattering are used to track the structure formation of BHJ thin films formed from the donor polymer poly(2,5‐bis(3‐hexadecylthiophen‐2‐yl)thieno[3,2‐b]thiophene) with different fullerene derivative acceptors. The formation of stable bimolecular crystals through the intercalation of fullerene molecules between the side chains of polymer crystallites is investigated. Such systems exhibit more efficient exciton dissociation but lower photo‐conductance and faster decay of charges. On the basis of the experimental observations, intercalation obviously takes place before or with the formation of the crystalline polymer domains. It results in more stable structures whose volume remains constant upon further drying. Three distinct periods of drying are observed and the formation of unidimensional fullerene channels along the π‐stacking direction of polymer crystallites is confirmed.     

Effects of Central Corneal Stromal Thickness and Epithelial Thickness on Intraocular Pressure Using Goldmann Applanation and Non-Contact Tonometers

Purpose

To investigate whether corneal thickness parameters measured by optical coherence tomography (OCT), such as central corneal thickness (CCT), central corneal stromal thickness (CCST), and central corneal epithelial thickness (CCET), influence the intraocular pressure (IOP) difference measured by Goldmann applanation tonometry (GAT) and non-contact tonometry (NCT).

Methods

In total, 50 eyes from 50 subjects without glaucomatous defects were included in this retrospective, cross-sectional study. We measured IOP using GAT and NCT and calculated the difference between the two methods. CCT was measured by a Cirrus HD-OCT device using anterior segment imaging. The basement membrane of the epithelium, which was seen as a high-reflection line in the OCT image, was taken as a reference line to measure CCST and CCET.

Results

The mean IOP measured by GAT and NCT was 16.7 ± 3.0 and 18.1 ± 3.8 mmHg, respectively. The mean IOP difference was 1.5 ± 1.7 mmHg, and the IOP measured by NCT was 8.4% ± 11.3% higher than that measured by GAT. The CCET and CCST were 57.9 ± 5.6 and 501.7 ± 33.8 μm, respectively. CCT showed a positive correlation with both GAT IOP (r = 0.648, P < 0.001) and NCT IOP (r = 0.676, P < 0.001). Although CCST showed a significant correlation with GAT IOP and NCT IOP, CCET did not. The difference between GAT IOP and NCT IOP increased with CCT (r = 0.333, P = 0.018), and CCET was positively correlated with the IOP difference between GAT and NCT (r = 0.435, P = 0.002).

Conclusions

IOP increased with greater CCT, and CCST seemed to have a more important role than CCET. CCET also increased with greater CCT, and this may be a possible explanation for the increasing difference in IOP between GAT and NCT with increasing CCT.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

Conserved metabolic enzymes as vaccine antigens for giardiasis

Giardia lamblia is a leading protozoal cause of diarrheal disease worldwide. Infection is associated with abdominal pain, malabsorption and weight loss, and protracted post-infectious syndromes. A human vaccine is not available against G. lamblia. Prior studies with human and murine immune sera have identified several parasite antigens, including surface proteins and metabolic enzymes with intracellular functions. While surface proteins have demonstrated vaccine potential, they can exhibit significant variation between G. lamblia strains. By comparison, metabolic enzymes show greater conservation but their vaccine potential has not been established. To determine whether such proteins can serve as vaccine candidates, we focused on two enzymes, α-enolase (ENO) and ornithine carbamoyl transferase (OCT), which are involved in glycolysis and arginine metabolism, respectively. We show in a cohort of patients with confirmed giardiasis that both enzymes are immunogenic. Intranasal immunization with either enzyme antigen in mice induced strong systemic IgG1 and IgG2b responses and modest mucosal IgA responses, and a marked 100- to 1,000-fold reduction in peak trophozoite load upon oral G. lamblia challenge. ENO immunization also reduced the extent and duration of cyst excretion. Examination of 44 cytokines showed only minimal intestinal changes in immunized mice, although a modest increase of CCL22 was observed in ENO-immunized mice. Spectral flow cytometry revealed increased numbers and activation state of CD4 T cells in the small intestine and an increase in α4β7-expressing CD4 T cells in mesenteric lymph nodes of ENO-immunized mice. Consistent with a key role of CD4 T cells, immunization of CD4-deficient and Rag-2 deficient mice failed to induce protection, whereas mice lacking IgA were fully protected by immunization, indicating that immunity was CD4 T cell-dependent but IgA-independent. These results demonstrate that conserved metabolic enzymes can be effective vaccine antigens for protection against G. lamblia infection, thereby expanding the repertoire of candidate antigens beyond primary surface proteins.