AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

Cutaneous Vasculopathy in a COVID-19 Critically Ill Patient: A Histologic, Immunohistochemical, and Electron Microscopy Study.

We describe a critically ill, SARS-CoV-2 positive patient with respiratory failure and thrombotic/livedoid skin lesions, appearing during the course of the disease. The biopsy of the lesions revealed an occlusive, pauci-inflammatory vasculopathy of the cutaneous small vessels characterized by complement and fibrinogen deposition on vascular walls, pointing to a thrombotic vasculopathy. Transmission electron microscopy of the affected skin failed to reveal any viral inclusions. Clinical evaluation and laboratory findings ruled out systemic coagulopathies and disseminated intravascular coagulation, drug-induced skin reaction, and common viral rashes. Our hypothesis is that the, herein evidenced, microvascular occlusive injury might constitute a significant pathologic mechanism in COVID-19, being a common denominator between cutaneous and pulmonary manifestations.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

Changes in the cellular fatty acid profile drive the proteasomal degradation of α-synuclein and enhance neuronal survival.

Parkinson's disease is biochemically characterized by the deposition of aberrant aggregated α-synuclein in the affected neurons. The aggregation properties of α-synuclein greatly depend on its affinity to bind cellular membranes via a dynamic interaction with specific lipid moieties. In particular, α-synuclein can interact with arachidonic acid (AA), a polyunsaturated fatty acid, in a manner that promotes the formation of α-helix enriched assemblies. In a cellular context, AA is released from membrane phospholipids by phospholipase A2 (PLA2 ). To investigate the impact of PLA2 activity on α-synuclein aggregation, we have applied selective PLA2 inhibitors to a SH-SY5Y cellular model where the expression of human wild-type α-synuclein is correlated with a gradual accumulation of soluble oligomers and subsequent cell death. We have found that pharmacological and genetic inhibition of GIVA cPLA2 resulted in a dramatic decrease of intracellular oligomeric and monomeric α-synuclein significantly promoting cell survival. Our data suggest that alterations in the levels of free fatty acids, and especially AA and adrenic acid, promote the formation of α-synuclein conformers which are more susceptible to proteasomal degradation. This mechanism is active only in living cells and is generic since it does not depend on the absolute quantity of α-synuclein, the presence of disease-linked point mutations, the expression system or the type of cells. Our findings indicate that the α-synuclein-fatty acid interaction can be a critical determinant of the conformation and fate of α-synuclein in the cell interior and, as such, cPLA2 inhibitors could serve to alleviate the intracellular, potentially pathological, α-synuclein burden.

Increased autophagy/mitophagy levels in primary tumours of patients with pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS: We assessed the levels of autophagy and mitophagy, that are linked to cancer development and drug resistance, in well differentiated pancreatic neuroendocrine neoplasms (PanNENs) and correlated them with clinico-pathological parameters. METHODS: Fluorescent immunostaining for the autophagy markers LC3Β and p62/or LAMP1 was performed on 22 PanNENs and 11 controls of normal pancreatic tissues and validated through Western blotting. Autophagy quantitative scoring was generated for LC3B-positive puncta and analysed in relation to clinico-pathological parameters. TOMM20/LC3B qualitative assessment of mitophagy levels was undertaken by fluorescent immunostaining. The presence of autophagy/mitophagy was validated by transmission electron microscopy. RESULTS: Autophagy levels (LC3B-positive puncta/cell) were discriminative for normal vs. NEN pancreatic tissue (p = 0.007). A significant association was observed between autophagy levels and tumour grade (Ki67 < 3% vs. Ki67 ≥ 3%; p = 0.021), but not functionality (p = 0.266) size (cut-off of 20 mm; p = 0.808), local invasion (p = 0.481), lymph node- (p = 0.849) and distant metastases (p = 0.699). Qualitative assessment of TOMM20/LC3B demonstrated strong mitophagy levels in PanNENs by fluorescent immunostaining as compared with normal tissue. Transmission electron microscopy revealed enhanced autophagy and mitophagy in PanNEN tissue. Response to molecular targeted therapies in metastatic cases (n = 4) did not reveal any patterns of association to autophagy levels. CONCLUSIONS: Increased autophagy levels are present in primary tumours of patients with PanNENs and are partially attributed to upregulated mitophagy. Grade was the only clinico-pathological parameter associated with autophagy scores.

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats.

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.