Cellular Prion Protein Is Essential for Myocardial Regeneration but Not the Recovery of Left Ventricular Function from Apical Ballooning

This study tested the hypothesis that cellular prion protein (PrPC) played an essential role in myocardial regeneration and recovery of left ventricular ejection fraction (LVEF) from apical takotsubo cardiomyopathy (TCM) induced by transaortic constriction (TAC). In vitro study was categorized into G1 (H9C2), G2 (H9C2-overexpression-PrPC), G3 (H9C2-overexpression-PrPC + Stelazine/1 uM), and G4 (H9C2 + siRNA-PrPC), respectively. The results showed that the protein expressions of PrPC, cell-stress signaling (p-PI3K/p-Akt/p-m-TOR) and signal transduction pathway for cell proliferation/division (RAS/c-RAF/p-MEK/p-ERK1/2) were lowest in G1, highest in G2, significantly higher in G3 than in G4 (all p < 0.001). Adult-male B6 mice (n = 30) were equally categorized in group 1 (sham-control), group 2 (TAC) for 14 days, then relieved the knot and administered BrdU (50 ug/kg/intravenously/q.6.h for two times from day-14 after TAC) and group 3 (TAC + Stelazine/20 mg/kg/day since day 7 after TAC up to day 21 + BrdU administered as group 2), and animals were euthanized at day 28. The results showed that by day 28, the LVEF was significantly higher in group 1 than in groups 2/3 and significantly higher in group 3 than in group 2, whereas the LV chamber size exhibited an opposite pattern of LVEF (all p < 0.0001). The protein expressions of PrPC/p-PI3K/p-Akt/p-m-TOR/cyclin D/cyclin E and cellular-proliferation biomarkers (Ki67/PCNA/BrdU) exhibited an opposite pattern of LVEF (all p < 0.0001) among the three groups, whereas the protein expressions of RAS/c-RAF/p-MEK/p-ERK1/2 were significantly and progressively increased from groups 1 to 3 (all p < 0.0001). In conclusion, PrPC participated in regulating the intrinsic response of cell-stress signaling and myocardial regeneration but did not offer significant benefit on recovery of the heart function in the setting of TCM.

A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes

Background: Activation of stress pathways intrinsic to the β cell are thought to both accelerate β cell death and increase β cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking. Methods: To identify temporal and disease-related changes in islet β cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. Findings: In islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of β cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, β cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls. Interpretation: We identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of β cell stress in T1D.

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress abrogated Dragotcytosis frequency, that ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH and oxidative stresses and correlated with the macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

Update on Myositis Therapy: from Today’s Standards to Tomorrow’s Possibilities

: Inflammatory myopathies, in short, myositis, are heterogeneous disorders that are characterized by inflammation of skeletal muscle and weakness of arms and legs. Research over the past few years has led to a new understanding regarding the pathogenesis of myositis. The new insights include different pathways of the innate and adaptive immune response during the pathogenesis of myositis. The importance of non-inflammatory mechanisms such as cell stress and impaired autophagy has been recently described. New target-specific drugs for myositis have been developed and are currently being tested in clinical trials. In this review, we discuss the mechanisms of action of pharmacological standards in myositis and provide an outlook of future treatment approaches.

EXTH-25. ENHANCING GLIOBLASTOMA CELL STRESS RESPONSE TO IMPROVE GAMMA DELTA T-CELL IMMUNOTHERAPY

Glioblastoma (GBM) is an aggressive cancer that has been largely intractable to novel therapies, however, enhancing the efficacy of immunotherapy could potentially overcome immunosuppression and potentially improve patient outcomes. The cellular stress induced by Temozolomide (TMZ) increases innate immune ligands, which could be exploited to promote immune recognition. TMZ-induced DNA damage can activate the stress response pathway, increasing the expression of NKG2D ligands (NKG2DL) on tumor cells. This leads to an increase in NKG2DL recognition by NKG2D receptors on both natural killer and cytotoxic T-cells to elicit a cytotoxic effect. The lymphodepleting effect of TMZ, however, can limit the ability of these cells to recognize and kill tumor cells. TMZ was shown to induce NKG2DL in gliomas both in vitro and in vivo, providing the basis for clinical trials of TMZ in combination with genetically engineered TMZ-resistant gamma delta T-cells (NCT04165941). To further promote immune recognition, we sought to augment the TMZ-induced stress response by exploring the combination of DNA alkylation with either PARP (Niraparib) or ATM Kinase inhibition (AZD1390). Combinatorial therapy significantly, but heterogeneously, increased differential subsets of NKG2DL genes in comparison to TMZ alone in GBM cells isolated from patient derived xenografts (PDX): 1) MICA and MICB were increased at least 10-fold in D456 (proneural) cells; 2) ULBP1 and ULBP2 were increased at least 2-fold in JX39 (classical) cells; and 3) minimal increases in NKG2DLs were observed in JX22 (mesenchymal) cells. Repression of NKG2DLs by hypoxia/low glucose was also heterogeneous, being observed in two of three GBM models tested. We are currently determining whether these combinatorial treatments improve gamma delta T-cell cytotoxicity against GBM cells and in vivo tumor models. Taken together, our data suggest that enhancing cell stress responses induced by chemotherapies may permit novel immunotherapy therapeutic interventions for brain tumor patients.

ATG8ylation of proteins: A way to cope with cell stress?

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed “ATG8ylation,” which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers.

Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy

The expression of the calcitonin receptor (CT Receptor) is widespread throughout the life cycle of mammals and in many diseases, and in these contexts the functions of the common isoforms is largely unknown. The relatively recent development of anti-CT Receptor antibodies that bind separate epitopes on the CTa Receptor and CTb Receptor isoforms has advanced our knowledge and understanding of these events. CT Receptor at the protein level is upregulated in programmed cell death including apoptosis (as described in a previous publication) and autophagy, which is discussed in our upcoming, unpublished review. Incomplete data sets are cited in this review on the upregulation of CACLR (encoding CT Receptor) mRNA, in particular the insert-positive isoform (CTb Receptor), in response to cell stress. Cell stress is induced by growth in depleted foetal bovine serum (dFBS) or without FBS, both of which induce degrees of starvation and autophagy, or dFBS plus staurosporine, which induces apoptosis. Details of the methods deployed to generate these data are described here including measurement of the upregulation of CTb Receptor mRNA with qPCR and nanopore long range sequencing. An anti-CT Receptor antibody also known as CalRexinTM, which binds an epitope in the N-terminal domain, was conjugated to either fluorophore 568, which is accumulated into apoptotic cells as previously reported, or pHrodo Red, a pH dependent fluorescent dye, which is accumulated into autophagic and apoptotic cells. These conjugates are under development to image programmed cell death. The methods for conjugation and high content imaging on the Operetta platform are described. The high fluorescence intensity at low pH of CalRexin:pHrodo Red in both autophagic and apoptotic cells suggests localisation in autophago-lysosomes and lysosomes respectively. Overall, these observations and the methods that underpin them have contributed to our understanding of the widespread expression of CT Receptor isoforms.