Inhibition of the extracellular signal-regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells.

Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-α and the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.

Endoplasmic Reticulum (ER) and ER-Phagy.

The endoplasmic reticulum (ER) is a biosynthetic organelle in eukaryotic cells. Its capacity to produce proteins, lipids and oligosaccharides responds to physiologic and pathologic demand. The transcriptional and translational unfolded protein response (UPR) programs increase ER size and activity. In contrast, ER-phagy programs in all their flavors select ER subdomains for lysosomal clearance. These programs are activated by nutrient deprivation, accumulation of excess ER (recov-ER-phagy), production of misfolded proteins that cannot be degraded by ER-associated degradation and that are removed from cells by the so-called ER-to-lysosome-associated degradation (ERLAD). Selection of ER subdomains to be cleared from cells relies on ER-phagy receptors, a class of membrane-bound proteins displaying cytosolic domains that engage the cytosolic ubiquitin-like protein LC3. Mechanistically, ER clearance proceeds via macro-ER-phagy, micro-ER-phagy and LC3-regulated vesicular delivery.

Deep learning approach for quantification of organelles and misfolded polypeptide delivery within degradative compartments.

Endolysosomal compartments maintain cellular fitness by clearing dysfunctional organelles and proteins from cells. Modulation of their activity offers therapeutic opportunities. Quantification of cargo delivery to and/or accumulation within endolysosomes is instrumental for characterizing lysosome-driven pathways at the molecular level and monitoring consequences of genetic or environmental modifications. Here we introduce LysoQuant, a deep learning approach for segmentation and classification of fluorescence images capturing cargo delivery within endolysosomes for clearance. LysoQuant is trained for unbiased and rapid recognition with human-level accuracy, and the pipeline informs on a series of quantitative parameters such as endolysosome number, size, shape, position within cells, and occupancy, which report on activity of lysosome-driven pathways. In our selected examples, LysoQuant successfully determines the magnitude of mechanistically distinct catabolic pathways that ensure lysosomal clearance of a model organelle, the endoplasmic reticulum, and of a model protein, polymerogenic ATZ. It does so with accuracy and velocity compatible with those of high-throughput analyses.

Endoplasmic Reticulum and Lysosomal Quality Control of Four Nonsense Mutants of Iduronate 2-Sulfatase Linked to Hunter's Syndrome.

Hunter's syndrome (mucopolysaccharidosis type II) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene. Motivated by the case of a child affected by this syndrome, we compared the intracellular fate of wild-type IDS (IDSWT) and four nonsense mutations of IDS (IDSL482X, IDSY452X, IDSR443X, and IDSW337X) generating progressively shorter forms of IDS associated with mild to severe forms of the disease. Our analyses revealed formylation of all forms of IDS at cysteine 84, which is a prerequisite for enzymatic activity. After formylation, IDSWT was transported within lysosomes, where it was processed in the mature form of the enzyme. The length of disease-causing deletions correlated with gravity of the folding and transport phenotype, which was anticipated by molecular dynamics analyses. The shortest form of IDS, IDSW337X, was retained in the endoplasmic reticulum (ER) and degraded by the ubiquitin-proteasome system. IDSR443X, IDSY452X, and IDSL482X passed ER quality control and were transported to the lysosomes, but failed lysosomal quality control, resulting in their rapid clearance and in loss-of-function phenotype. Failure of ER quality control inspection is an established cause of loss of function observed in protein misfolding diseases. Our data reveal that fulfillment of ER requirements might not be sufficient, highlight lysosomal quality control as the distal station to control lysosomal enzymes fitness and pave the way for alternative therapeutic interventions.

A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.