The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions

Abstract

Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be anatomically segregated) co-exist and they are likely to interact at molecular level. In fact, LC3 and P20S co-immunoprecipitate, suggesting a specific binding and functional interplay, which may be altered by inhibiting mTOR. In summary, ATG and UP often represent two facets of a single organelle, in which unexpected amount of enzymatic activity should be available. Thus, autophagoproteasome may represent a sophisticated ultimate clearing apparatus.

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

“…Cells were then collected by centrifugation at 10,000 rpm for 10 min at 4°C to form a buffy coat of cells, washed with PBS and post-fixed in 1% OsO 4 for 1h at 4°C to prevent the formation of membranous artifacts, which may mimic autophagic vacuoles in TEM analyses [34]. Samples were dehydrated in ethanol and finally embedded in epoxy resin as previously described [35], to maintain the finest cell ultrastructure and to preserve epitopes for immunocytochemistry. Ultrathin sections (40-50 nm) were used for post-embedding immune-electron microscopy [35].…”

“…Samples were dehydrated in ethanol and finally embedded in epoxy resin as previously described [35], to maintain the finest cell ultrastructure and to preserve epitopes for immunocytochemistry. Ultrathin sections (40-50 nm) were used for post-embedding immune-electron microscopy [35]. Briefly, sections were collected on nickel grids and incubated in aqueous saturated NaIO 4 for 30 min at RT.…”

“…We scored as ATG-like vacuoles both double or multiple membranes (autophagosomes-like) containing cytoplasmic material and electron-dense membranous structures [35] A total number of 30 cells per experimental group was observed in non-serial sections of each grid. TDP-43-and P-TDP43-positive immune-gold particles were counted both in the nucleus and in the cytosol.…”

Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease

“…This concept does not rule out previous findings showing both proteasome and autophagosome dysfunction. In fact all these compartments eventually merge to produce an ultimate organelle which fuses with lysosomes (Lenzi et al, 2016). The specific fate of endosomes is related to the chance that, despite routinely shuttling toward the trans Golgi network, this compartment may be delivered to the cell membrane to be released.…”

Endogenous 3-Iodothyronamine (T1AM) and Synthetic Thyronamine-Like Analog SG-2 Act as Novel Pleiotropic Neuroprotective Agents through the Modulation of SIRT6

“…The present investigation was further motivated by recent findings, which indicate that the two major clearing pathways, namely autophagy and ubiquitine proteasome (UP), considered as independent for long time, converge at the level of single organelles named autophagoproteasomes following mTOR inhibition. Such a merging when considered at functional level might empower protein homeostasis and cell clearing [15,16]. In line with this evidence, we included an experimental part where the merging between autophagy and UP was investigated under the effects of T1AM and thyronamine-like analogs SG-1 and SG-2.…”