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Protein aggregates and cytoplasmic vacuolization are major hallmarks of multisystem proteinopathies (MSPs) that lead to muscle weakness. Here, we identify METTL21C as a skeletal muscle-specific lysine methyltransferase. Insertion of a β-galactosidase cassette into the Mettl21c mouse locus revealed that METTL21C is specifically expressed in MYH7-positive skeletal muscle fibers. Ablation of the Mettl21c gene reduced endurance capacity and led to age-dependent accumulation of autophagic vacuoles in skeletal muscle. Denervation-induced muscle atrophy highlighted further impairments of autophagy-related proteins, including LC3, p62, and cathepsins, in Mettl21c−/− muscles. In addition, we demonstrate that METTL21C interacts with the ATPase p97 (VCP), which is mutated in various human MSP conditions. We reveal that METTL21C trimethylates p97 on the Lys315 residue and found that loss of this modification reduced p97 hexamer formation and ATPase activity in vivo. We conclude that the methyltransferase METTL21C is an important modulator of protein degradation in skeletal muscle under both normal and enhanced protein breakdown conditions.
Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.
Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice
(2015)
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.
The extracellular matrix is rapidly emerging as a prominent contributor to various fundamental processes of tumorigenesis. In particular, decorin, a member of the small leucine-rich proteoglycan gene family, is assuming a central role as a potent soluble tumor repressor. Decorin binds and antagonizes various receptor tyrosine kinases and inhibits downstream oncogenic signaling in several solid tumors. Among other functions, decorin evokes cell cycle arrest, apoptosis, and antimetastatic, and antiangiogenic programs. Recent work has revealed a paradigmatic shift in our understanding of the molecular mechanisms underlying its tumoricidal properties. Decorin adversely compromises the genetic signature of the tumor microenvironment and induces endothelial cell autophagy downstream of VEGFR2. Moreover, decorin selectively evokes destruction of tumor cell mitochondria downstream of Met through mitophagy. Acting as a partial agonist, decorin signals via proautophagic receptors and triggers procatabolic processes that parallel the classical tumoricidal properties of this multifaceted proteoglycan.
Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.
Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)-assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct anti-tumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.
Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
Inflammation is a highly regulated biological response of the immune system that is triggered by assaulting pathogens or endogenous alarmins. It is now well established that some soluble extracellular matrix constituents, such as small leucine-rich proteoglycans (SLRPs), can act as danger signals and trigger aseptic inflammation by interacting with innate immune receptors. SLRP inflammatory signaling cascade goes far beyond its canonical function. By choosing specific innate immune receptors, coreceptors, and adaptor molecules, SLRPs promote a switch between pro- and anti-inflammatory signaling, thereby determining disease resolution or chronification. Moreover, by orchestrating signaling through various receptors, SLRPs fine-tune inflammation and, despite their structural homology, regulate inflammatory processes in a molecule-specific manner. Hence, the overarching theme of this review is to highlight the molecular and functional specificity of biglycan-, decorin-, lumican-, and fibromodulin-mediated signaling in inflammatory and autoimmune diseases.
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
As current classical Hodgkin lymphoma (cHL) treatment strategies have pronounced side-effects, specific inhibition of signaling pathways may offer novel strategies in cHL therapy. Basal autophagy, a regulated catabolic pathway to degrade cell's own components, is in cancer linked with both, tumor suppression or promotion. The finding that basal autophagy enhances tumor cell survival would thus lead to immediately testable strategies for novel therapies. Thus, we studied its contribution in cHL.We found constitutive activation of autophagy in cHL cell lines and primary tissue. The expression of key autophagy-relevant proteins (e.g. Beclin-1, ULK1) and LC3 processing was increased in cHL cells, even in lymphoma cases. Consistently, cHL cells exhibited elevated numbers of autophagic vacuoles and intact autophagic flux. Autophagy inhibition with chloroquine or inactivation of ATG5 induced apoptosis and reduced proliferation of cHL cells. Chloroquine-mediated inhibition of basal autophagy significantly impaired HL growth in-vivo in NOD SCID γc-/- (NSG) mice. We found that basal autophagy plays a pivotal role in sustaining mitochondrial function.We conclude that cHL cells require basal autophagy for growth, survival and sustained metabolism making them sensitive to autophagy inhibition. This suggests basal autophagy as useful target for new strategies in cHL treatment.