Chaperone-assisted Selective Autophagy Research Articles

Synaptopodin-2 Isoforms Have Specific Binding Partners and Display Distinct, Muscle Cell Type-Specific Expression Patterns.

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.

High-intensity interval training alleviates exhaustive exercise-induced HSP70-assisted selective autophagy in skeletal muscle

This study was designed to probe the effect of chaperone-assisted selective autophagy (CASA) on the maintenance of proteostasis during exhaustive exercise and uncover the alteration of CASA in muscle fibers with pre-high-intensity interval training (HIIT) intervention-induced muscle adaptation in response to exhaustive exercise. Rats were randomly divided into a control group; an exhaustive exercise group; and an HIIT + exhaustive exercise group. Results show myofibril damage and BiP levels were increased after exhaustive exercise, and the levels of the HSP70, BAG3, ubiquitin, autophagy-related proteins, and their interactions were increased. HIIT intervention before exhaustive exercise could decrease myofibril injury and BiP levels, accompanied by down-regulation of HSP70/BAG3 complex and selective autophagy. In conclusion, exhaustive exercise promotes CASA to clear protein aggregation for keeping proteostasis in muscle fibers; pre-HIIT intervention improves myofibril injury and unfold protein response caused by exhaustive exercise, which might contribute to inhibit the augmentation of CASA.

UPR-induced Vigorous Chaperone-assisted Selective Autophagy In Muscle Fiber During Exhaustive Exercise Was Improved By HIIT

UPR-induced Vigorous Chaperone-assisted Selective Autophagy In Muscle Fiber During Exhaustive Exercise Was Improved By HIIT

Abstract P2112: Mechanism Of Bag3's Degradation Of Tgfbr2 In Cardiac Fibroblasts

BAG3 levels are diminished in patients with heart failure and several mutations have been identified to cause dilated cardiomyopathy with cardiac fibrosis. BAG3’s four binding domains are critical to its function to facilitate proteostasis. BAG3’s role has been extensively studied in CMs, where it aids sarcomere protein turnover through chaperone-assisted selective autophagy, through BAG3’s complex with HSP70, HSPB8, and E3 ligase CHIP. However, BAG3 is ubiquitously expressed and its role in non-myocytes has been overlooked. Cardiac fibroblasts are responsible for the excessive matrix deposition leading to fibrosis, mostly through TGF-beta signaling. Previously, we have shown the importance of BAG3 in cardiac fibroblasts through its role in TGF-beta signaling and identified BAG3 to facilitate the degradation of the type II TGF-beta receptor (TGFBR2). As BAG3 does not have inherent E3 activity, this study aimed to identify the specific E3 ligase of TGFBR2 and the binding domains of BAG3 that are crucial to this interaction to gain insight into the effects of the clinical mutations of BAG3. Immunoprecipitation (IP) of endogenous BAG3 in cardiac fibroblasts revealed five E3 ligases as high-confidence interactors of BAG3. To find the specific E3 ligase of TGFBR2, we performed an IP of a V5-tagged TGFBR2 and identified HSP70, HSPB8 and CHIP as binding partners. To delineate the critical domains of BAG3 that impact the TGFBR2 degradation, we overexpressed 3x-FLAG-tagged BAG3 proteins lacking each domain respectively (WW, IPV, PxxP, and BAG) and show that the BAG domain is imperative in TGFBR2 degradation. This has clinical implications for the common DCM-causing E455K mutation in the BAG domain. The ubiquitination of TGFBR2 was decreased upon overexpression of a BAG3 plasmid containing the E455K mutation. Lastly, the drug JG-98, which disrupts the binding of HSP70 to BAG3, was used to show the importance of the HSP70-BAG3 axis in the degradation of TGFBR2. In conclusion, we have uncovered the molecular mechanism of TGFBR2 degradation in cardiac fibroblasts mediated by the BAG3-HSP70-HSPB8 complex, with clinical implications for the loss of BAG3 and several common DCM-causing mutations in the development of cardiac fibrosis.

Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells andplays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway byknocking down Bag3 can promote desmin aggregate formation.

Novel Filamin C Myofibrillar Myopathy Variants Cause Different Pathomechanisms and Alterations in Protein Quality Systems.

Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.

Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis.

Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction.

HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies

ABSTRACT Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases. Abbreviations: ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Chaperone-assisted selective autophagy targets filovirus VP40 as a client and restricts egress of virus particles.

The filovirus VP40 protein directs virion egress, which is regulated either positively or negatively by select VP40-host interactions. We demonstrate that host BAG3 and HSP70 recognize VP40 as a client and inhibit the egress of VP40 virus-like particles (VLPs) by promoting degradation of VP40 via Chaperone-assisted selective autophagy (CASA). Pharmacological inhibition of either the early stage formation of the VP40/BAG3/HSP70 tripartite complex, or late stage formation of autolysosomes, rescued VP40 VLP egress back to WT levels. The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of autophagy, and we found that surface expression of EBOV GP on either VLPs or an infectious VSV recombinant virus, activated mTORC1. Notably, pharmacological suppression of mTORC1 signaling by rapamycin activated CASA in a BAG3-dependent manner to restrict the egress of both VLPs and infectious EBOV in Huh7 cells. In sum, our findings highlight the involvement of the mTORC1/CASA axis in regulating filovirus egress.

Identification of HSPB8 modulators counteracting misfolded protein accumulation in neurodegenerative diseases

AimsThe small Heat Shock Protein B8 (HSPB8) is the core component of the Chaperone-Assisted Selective Autophagy (CASA) complex. This complex selectively targets, transports, and tags misfolded proteins for their recognition by autophagy receptors and insertion into the autophagosome for clearance. CASA is essential to maintain intracellular proteostasis, especially in heart, muscle, and brain often exposed to various types of cell stresses. In neurons, HSPB8 protects against neurotoxicity caused by misfolded proteins in several models of neurodegenerative diseases; by facilitating autophagy, HSPB8 assists misfolded proteins degradation also counteracting proteasome overwhelming and inhibition. Materials and methodsTo enhance HSPB8 protective activity, we screened a library of approximately 120,000 small molecules to identify compounds capable of increasing HSPB8 gene transcription, translation, or protein stability. Key findingsWe found 83 active compounds active in preliminary dose-response assays and further classified them in 19 chemical classes by medicinal chemists' visual inspection. Of these 19 prototypes, 14 induced HSPB8 mRNA and protein levels in SH-SY5Y cells. Out of these 14 compounds, 3 successfully reduced the aggregation propensity of a disease-associated mutant misfolded superoxide dismutase 1 (SOD1) protein in a flow cytometry-based aggregation assay (Flow cytometric analysis of Inclusions and Trafficking (FloIT)) and induced the expression (mRNA and protein) of some autophagy receptors. Notably, the 3 hits were inactive in HSPB8-depleted cells, confirming that their protective activity is mediated by and requires HSPB8. SignificanceThese compounds may be highly relevant for a therapeutic approach in several human disorders, including neurodegenerative diseases, in which enhancement of CASA exerts beneficial activities.

Silica Nanoparticles Trigger Chaperone HSPB8-Assisted Selective Autophagy via TFEB Activation in Hepatocytes.

Silica nanoparticles (SiNPs) are one of the most common inorganic nanomaterials. Autophagy is the predominant biological response to nanoparticles and transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway. Previous studies show that SiNPs induce autophagosome accumulation, yet the precise underlying mechanisms remain uncertain. The present study investigates the role of TFEB during SiNP-induced autophagy. SiNP-induced TFEB nuclear translocation is verified using immunofluorescence and western blot assay. The regulation of TFEB is proved to be via EIF2AK3 pathway. A TFEB knockout (KO) cell line is constructed to validate the TFEB involvement in SiNP-induced autophagy. The transcriptomes of wild-type and TFEB KO cells are compared using RNA-sequencing to identify genes of the TFEB-mediated autophagy and lysosome pathways affected by SiNPs. Based on these data and the Human Autophagy Database, four candidate autophagic genes are identified, including HSPB8, ATG4D, CTSB and CTSD. Specifically, that the chaperone HSPB8 is upregulated through SiNP-mediated TFEB activation and forms a chaperone-assisted selective autophagy (CASA) complex with BAG3 and HSC70, triggering HSPB8-assisted selective autophagy, is found. Thus, this study characterizes a novel mechanism underlying SiNP-induced autophagy that helps pave the way for further research on the toxicity and risk assessment of SiNPs.

HSPB8 counteracts tumor activity of BRAF- and NRAS-mutant melanoma cells by modulation of RAS-prenylation and autophagy

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60–70% and 15–20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

BAG3 Alleviates Atherosclerosis by Inhibiting Endothelial-to-Mesenchymal Transition via Autophagy Activation.

Atherosclerosis is a chronic systemic inflammatory disease that causes severe cardiovascular events. B cell lymphoma 2-associated athanogene (BAG3) was proven to participate in the regulation of tumor angiogenesis, neurodegenerative diseases, and cardiac diseases, but its role in atherosclerosis remains unclear. Here, we aim to investigate the role of BAG3 in atherosclerosis and elucidate the potential molecular mechanism. In this study, ApoE−/− mice were given a tail-vein injection of BAG3-overexpressing lentivirus and fed a 12-week high-fat diet (HFD) to investigate the role of BAG3 in atherosclerosis. The overexpression of BAG3 reduced plaque areas and improved atherosclerosis in ApoE−/− mice. Our research proves that BAG3 promotes autophagy in vitro, contributing to the suppression of EndMT in human umbilical vein endothelial cells (HUVECs). Mechanically, autophagy activation is mediated by BAG3 via the interaction between BAG3 and its chaperones HSP70 and HSPB8. In conclusion, BAG3 facilitates autophagy activation via the formation of the chaperone-assisted selective autophagy (CASA) complex interacting with HSP70 and HSPB8, leading to the inhibition of EndMT during the progression of atherosclerosis and indicating that BAG3 is a potential therapeutic target for atherosclerosis.

The role of BAG3 in dilated cardiomyopathy and its association with Charcot-Marie-Tooth disease type 2.

Bcl2-associated athanogene 3 (BAG3) is a multifunctional cochaperone responsible for protein quality control within cells. BAG3 interacts with chaperones HSPB8 and Hsp70 to transport misfolded proteins to the Microtubule Organizing Center (MTOC) and degrade them in autophagosomes in a process known as Chaperone Assisted Selective Autophagy (CASA). Mutations in the second conserved IPV motif of BAG3 are known to cause Dilated Cardiomyopathy (DCM) by inhibiting adequate removal of non-native proteins. The proline 209 to leucine (P209L) BAG3 mutant in particular causes the aggregation of BAG3 and misfolded proteins as well as the sequestration of essential chaperones. The exact mechanisms of protein aggregation in DCM are unknown. However, the similar presence of insoluble protein aggregates in Charcot-Marie-Tooth disease type 2 (CMT2) induced by the proline 182 to leucine (P182L) HSPB1 mutant points to a possible avenue for future research: IPV motif. In this review, we summarize the molecular mechanisms of CASA and the currently known pathological effects of mutated BAG3 in DCM. Additionally, we will provide insight on the importance of the IPV motif in protein aggregation by analyzing a potential association between DCM and CMT2.

ERAS Is Constitutively Expressed in the Tissues of Adult Horses and May Be a Key Player in Basal Autophagy.

ERas is a new gene of the Ras family found in murine embryonic stem (ES) cells. Its human ortholog is not expressed in human ES cells. So far ERas gene has only been found to be expressed in the tissues of adult cynomolgus monkeys and cattle; however, information about ERAS expression or its potential functions in equine tissues is lacking. This study was performed to investigate whether Eras is an equine functional gene and whether ERAS is expressed in the tissues of adult horses and determine its potential physiological role. Expression of the ERas gene was detected in all examined adult tissues, and the RT-PCR assay revealed ERAS transcripts. Protein expression was also detected by Western blot analysis. Quantitative real time RT-qPCR analysis revealed that different expression levels of ERAS transcripts were most highly expressed in the testis. Immunohistochemically, ERAS was found to be localized prevalently in the plasmatic membrane as well as cytoplasm of the cells. ERAS was a physical partner of activated PDGFβR leading to the AKT signaling. ERAS was found to interact with a network of proteins (BAG3, CHIP, Hsc70/Hsp70, HspB8, Synpo2, and p62) known to play a role in the chaperone-assisted selective autophagy (CASA), which is also known as BAG3-mediated selective macroautophagy, an adaptive mechanism to maintain cellular homeostasis. Furthermore, ERAS was found to interact with parkin. PINK1, BNIP3, laforin. All these proteins are known to play a role in parkin-dependent and -independent mitophagy. This is the first study demonstrating that Eras is a functional gene, and that ERAS is constitutively expressed in the tissues of adult horses. ERAS appears to play a physiological role in cellular proteostasis maintenance, thus mitigating the proteotoxicity of accumulated misfolded proteins and contributing to protection against disease. Finally, it is conceivable that activation of AKT pathway by PDGFRs promotes actin reorganization, directed cell movements, stimulation of cell growth.

Neurodegenerative Disease-Associated TDP-43 Fragments Are Extracellularly Secreted with CASA Complex Proteins.

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs.

Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells.

ObjectiveThe periodontal ligament (PDL) is exposed to constant mechanical forces potentiated by orthodontic tooth movement (OTM). The aim of our study was to investigate the involvement of chaperone-assisted selective autophagy (CASA) in mechanosensing and cellular adaption to forces in the PDL.Materials and methodsHuman PDL cells were loaded with 2.5, 5, and 10% of static mechanical strain for 24 h in vitro. Untreated cells served as controls. Gene expression of HSPA8, HSPB8, BAG3, STUB1, SYNPO2 was investigated via RT-qPCR (Quantitative reverse transcription PCR). Western blot evidenced protein expression of these molecules and of Filamin A. In vivo analyses of CASA were performed via immunohistochemistry on teeth with and without OTM.ResultsCASA machinery genes were inherently expressed in PDL cells and exhibited transcriptional induction upon mechanical strain. Protein analyses underlined these findings, even though modulation upon force exertion also demonstrated a decrease for some molecules and loading strengths. In vivo results evidenced again the uniform upregulation of HSPA8, HSPB8, BAG3, STUB1, SYNPO2 and Filamin A in teeth with OTM compared to controls. Experiments generally evidenced a pronounced variability in the expression between donors both on the gene and protein level.ConclusionsOur study is the first to identify both the expression and functional relevance of CASA in the PDL. The data reflect its probable central role in adequate adaption to forces exerted by OTM and in mechanical stress protection of cells. Deeper knowledge of the CASA pathway will allow better assessment of predisposing factors regarding side effects during mechanical force application that can be used in orthodontic practice.

Strain-induced mechanoresponse depends on cell contractility and BAG3-mediated autophagy

Basically, all mammalian tissues are constantly exposed to a variety of environmental mechanical signals. Depending on the signal strength, mechanics intervenes in a multitude of cellular processes and is thus capable of inducing simple cellular adaptations but also complex differentiation processes and even apoptosis. The underlying recognition typically depends on mechanosensitive proteins, which most often sense the mechanical signal for the induction of a cellular signaling cascade by changing their protein conformation. However, the fate of mechanosensors after mechanical stress application is still poorly understood, and it remains unclear whether protein degradation pathways affect the mechanosensitivity of cells. Here, we show that cyclic stretch induces autophagosome formation in a time-dependent manner. Formation depends on the cochaperone BAG family molecular chaperone regulator 3 (BAG3) and thus likely involves BAG3-mediated chaperone-assisted selective autophagy. Furthermore, we demonstrate that strain-induced cell reorientation is clearly delayed upon inhibition of autophagy, suggesting a bidirectional cross-talk between mechanotransduction and autophagic degradation. The strength of the observed delay depends on stable adhesion structures and stress fiber formation in a Ras homologue family member A (RhoA)-dependent manner.

Updates on the Immunopathology in Idiopathic Inflammatory Myopathies.

To review recent advances in immunopathology for idiopathic inflammatory myopathies, focusing on widely available immunohistochemical analyses. Sarcoplasmic expression of myxovirus resistance protein A (MxA) is specifically observed in all types of dermatomyositis and informs that type I interferons are crucially involved in its pathogenesis. It is a more sensitive diagnostic marker than perifascicular atrophy. Diffuse tiny dots in the sarcoplasm highlighted by p62 immunostaining are characteristically seen in immune-mediated necrotizing myopathy. This feature is linked to a chaperone-assisted selective autophagy pathway. Myofiber invasion by highly differentiated T cells, a marker of which is KLRG1, is specific to inclusion body myositis and has a crucial role in its pathogenesis. The recent advances in immunopathology contribute to increased diagnostic accuracy and a better understanding of the underlying pathophysiology in different types of idiopathic inflammatory myopathies.