Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.

The Effect of Free and Protein-Bound Maillard Reaction Products N-ε-Carboxymethyllysine, N-ε-Fructosyllysine, and Pyrraline on Nrf2 and NFκB in HCT 116 Cells.

SCOPE: Maillard reaction products (MRPs) are believed to interact with the receptor for advanced glycation endproducts (RAGE) and lead to a pro-inflammatory cellular response. The structural basis for this interaction is scarcely understood. This study investigates the effect of individual lysine modifications in free form or bound to casein on human colon cancer cells. METHODS AND RESULTS: Selectively glycated casein containing either protein-bound N-ε-carboxymethyllysine (CML), N-ε-fructosyllysine (FL), or pyrraline is prepared and up to 94%, 97%, and 61% of lysine modification could be attributed to CML, FL, or pyrraline, respectively. HCT 116 cells are treated with free CML, pyrraline, FL, or modified casein for 24 h. Native casein is used as control. Intracellular MRP content is analyzed by UPLC-MS/MS. Microscopic analysis of the transcription factors shows no activation of NFκB by free or protein-bound FL or CML, whereas casein containing protein-bound pyrraline activates Nrf2. RAGE expression is not influenced by free or casein-bound MRPs. Activation of Nrf2 by pyrraline-modified casein is confirmed by analyzing Nrf2 target proteins NAD(P)H dehydrogenase (quinone 1) (NQO1) and heme oxygenase-1 (HO-1). CONCLUSION: Studies on the biological effects of glycated proteins require an individual consideration of defined structures. General statements on the effect of "AGEs" in biological systems are scientifically unsound.

The Alzheimer's disease-linked protease BACE1 modulates neuronal IL-6 signaling through shedding of the receptor gp130.

BACKGROUND: The protease BACE1 is a major drug target for Alzheimer's disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates. METHODS: To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors. RESULTS: Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal. CONCLUSION: BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.

A unified classification approach rating clinical utility of protein biomarkers across neurologic diseases.

A major evolution from purely clinical diagnoses to biomarker supported clinical diagnosing has been occurring over the past years in neurology. High-throughput methods, such as next-generation sequencing and mass spectrometry-based proteomics along with improved neuroimaging methods, are accelerating this development. This calls for a consensus framework that is broadly applicable and provides a spot-on overview of the clinical validity of novel biomarkers. We propose a harmonized terminology and a uniform concept that stratifies biomarkers according to clinical context of use and evidence levels, adapted from existing frameworks in oncology with a strong focus on (epi)genetic markers and treatment context. We demonstrate that this framework allows for a consistent assessment of clinical validity across disease entities and that sufficient evidence for many clinical applications of protein biomarkers is lacking. Our framework may help to identify promising biomarker candidates and classify their applications by clinical context, aiming for routine clinical use of (protein) biomarkers in neurology.

Experimental evidence for temporal uncoupling of brain Aß deposition and neurodegenerative sequelae.

Brain Aß deposition is a key early event in the pathogenesis of Alzheimer´s disease (AD), but the long presymptomatic phase and poor correlation between Aß deposition and clinical symptoms remain puzzling. To elucidate the dependency of downstream pathologies on Aß, we analyzed the trajectories of cerebral Aß accumulation, Aß seeding activity, and neurofilament light chain (NfL) in the CSF (a biomarker of neurodegeneration) in Aß-precursor protein transgenic mice. We find that Aß deposition increases linearly until it reaches an apparent plateau at a late age, while Aß seeding activity increases more rapidly and reaches a plateau earlier, coinciding with the onset of a robust increase of CSF NfL. Short-term inhibition of Aß generation in amyloid-laden mice reduced Aß deposition and associated glial changes, but failed to reduce Aß seeding activity, and CSF NfL continued to increase although at a slower pace. When short-term or long-term inhibition of Aß generation was started at pre-amyloid stages, CSF NfL did not increase despite some Aß deposition, microglial activation, and robust brain Aß seeding activity. A dissociation of Aß load and CSF NfL trajectories was also found in familial AD, consistent with the view that Aß aggregation is not kinetically coupled to neurotoxicity. Rather, neurodegeneration starts when Aß seeding activity is saturated and before Aß deposition reaches critical (half-maximal) levels, a phenomenon reminiscent of the two pathogenic phases in prion disease.

Signatures of glial activity can be detected in the CSF proteome.

Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. While these findings may eventually lead to new therapeutic opportunities, little is known about how these glial responses are reflected by biomarker changes in bodily fluids. Such knowledge, however, appears crucial for patient stratification, as well as monitoring disease progression and treatment responses in clinical trials. Here, we took advantage of well-described mouse models of ß-amyloidosis and α-synucleinopathy to explore cerebrospinal fluid (CSF) proteome changes related to their respective proteopathic lesions. Nontargeted liquid chromatography-mass spectrometry revealed that the majority of proteins that undergo age-related changes in CSF of either mouse model were linked to microglia and astrocytes. Specifically, we identified a panel of more than 20 glial-derived proteins that were increased in CSF of aged ß-amyloid precursor protein- and α-synuclein-transgenic mice and largely overlap with previously described disease-associated glial genes identified by single-cell transcriptomics. Our results also show that enhanced shedding is responsible for the increase of several of the identified glial CSF proteins as exemplified for TREM2. Notably, the vast majority of these proteins can also be quantified in human CSF and reveal changes in Alzheimer's disease cohorts. The finding that cellular transcriptome changes translate into corresponding changes of CSF proteins is of clinical relevance, supporting efforts to identify fluid biomarkers that reflect the various functional states of glial responses in cerebral proteopathies, such as Alzheimer's and Parkinson's disease.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates.

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-ß (Aß) peptides and formation of Aß deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aß pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aß1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aß deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

Beneficial Effect of ACI-24 Vaccination on Aß Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model.

Amyloid-ß (Aß) deposition is an initiating factor in Alzheimer's disease (AD). Microglia are the brain immune cells that surround and phagocytose Aß plaques, but their phagocytic capacity declines in AD. This is in agreement with studies that associate AD risk loci with genes regulating the phagocytic function of immune cells. Immunotherapies are currently pursued as strategies against AD and there are increased efforts to understand the role of the immune system in ameliorating AD pathology. Here, we evaluated the effect of the Aß targeting ACI-24 vaccine in reducing AD pathology in an amyloidosis mouse model. ACI-24 vaccination elicited a robust and sustained antibody response in APPPS1 mice with an accompanying reduction of Aß plaque load, Aß plaque-associated ApoE and dystrophic neurites as compared to non-vaccinated controls. Furthermore, an increased number of NLRP3-positive plaque-associated microglia was observed following ACI-24 vaccination. In contrast to this local microglial activation at Aß plaques, we observed a more ramified morphology of Aß plaque-distant microglia compared to non-vaccinated controls. Accordingly, bulk transcriptomic analysis revealed a trend towards the reduced expression of several disease-associated microglia (DAM) signatures that is in line with the reduced Aß plaque load triggered by ACI-24 vaccination. Our study demonstrates that administration of the Aß targeting vaccine ACI-24 reduces AD pathology, suggesting its use as a safe and cost-effective AD therapeutic intervention.

The pseudoprotease iRhom1 controls ectodomain shedding of membrane proteins in the nervous system.

Proteolytic ectodomain shedding of membrane proteins is a fundamental mechanism to control the communication between cells and their environment. A key protease for membrane protein shedding is ADAM17, which requires a non-proteolytic subunit, either inactive Rhomboid 1 (iRhom1) or iRhom2 for its activity. While iRhom1 and iRhom2 are co-expressed in most tissues and appear to have largely redundant functions, the brain is an organ with predominant expression of iRhom1. Yet, little is known about the spatio-temporal expression of iRhom1 in mammalian brain and about its function in controlling membrane protein shedding in the nervous system. Here, we demonstrate that iRhom1 is expressed in mouse brain from the prenatal stage to adulthood with a peak in early postnatal development. In the adult mouse brain iRhom1 was widely expressed, including in cortex, hippocampus, olfactory bulb, and cerebellum. Proteomic analysis of the secretome of primary neurons using the hiSPECS method and of cerebrospinal fluid, obtained from iRhom1-deficient and control mice, identified several membrane proteins that require iRhom1 for their shedding in vitro or in vivo. One of these proteins was 'multiple-EGF-like-domains protein 10' (MEGF10), a phagocytic receptor in the brain that is linked to the removal of amyloid ß and apoptotic neurons. MEGF10 was further validated as an ADAM17 substrate using ADAM17-deficient mouse embryonic fibroblasts. Taken together, this study discovers a role for iRhom1 in controlling membrane protein shedding in the mouse brain, establishes MEGF10 as an iRhom1-dependent ADAM17 substrate and demonstrates that iRhom1 is widely expressed in murine brain.

Highly efficient intercellular spreading of protein misfolding mediated by viral ligand-receptor interactions.

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.

Current concepts of ectopic nodal inclusions with special emphasis on nodal nevi.

Nodal inclusions of ectopic tissue within lymph nodes are seen comparatively often in dermatopathology and general pathology. Glandular and nonglandular epithelium, as well as melanocytic nevi can be observed within lymph nodes and represent mostly incidental findings without any relevance. The main challenge in reporting these morphologic features is to differentiate such benign inclusions from metastatic settlements of distinct organ tumors. As sentinel node biopsy and lymph node dissection have become standard procedure in clinical oncology and have an immense clinical impact, the correct evaluation of these nodal inclusions is indispensable to avoid undertreatment or overtreatment of patients. In addition, the genesis of these inclusions has not yet been satisfactorily clarified. Two concepts have been laid out: the theory of benign metastases and the migration arrest theory. However, neither theory has so far been able to answer the following questions: Why do we find more nodal nevi in patients with melanoma who had a sentinel node biopsy than in patients without melanoma, and why do we not find nodal nevi in deep visceral lymph nodes? We present a comprehensive review of the current knowledge on nodal inclusions, proposing a concept for the pathogenesis of nodal nevi, to answer these questions.

ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila.

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. We show that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. We find this stress adaptation to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. Our results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis.

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

Immunotherapy with NK cells: recent developments in gene modification open up new avenues.

Chimeric antigen receptor (CAR)-T cell therapies have achieved remarkable success. However, application-related toxicities, such as cytokine release syndrome or neurotoxicity, moved natural killer (NK) cells into focus as novel players in immunotherapy. CAR-NK cells provide an advantageous dual killing-capacity by CAR-dependent and -independent mechanisms and induce few side effects. While the majority of trials still use CAR-T cells, CAR-NK cell trials are on the rise with 19 ongoing studies worldwide. This review illuminates the current state of research and clinical application of CAR-NK cells, as well as future developmental potential.

Improved pre-hospital care efficiency due to the implementation of pre-hospital trauma life support (PHTLS®) algorithms.

PURPOSE: Pre-hospital trauma life support (PHTLS®) includes a standardized algorithm for pre-hospital care. Implementation of PHTLS® led to improved outcome in less developed medical trauma systems. We aimed to determine the impact of PHTLS® on quality of pre-hospital care in a European metropolitan area. We hypothesized that the introduction of PHTLS® was associated with improved efficiency of pre-hospital care for severely injured patients and less emergency physician deployment. METHODS: We included adult polytrauma (ISS > 15) patients that were admitted to our level one trauma center during a 7-year time period. Patients were grouped based on the presence or absence of a PHTLS®-trained paramedic in the pre-hospital trauma team. Group I (no-PHTLS group) included all casualties treated by no-PHTLS®-trained personnel. Group II (PHTLS group) was composed of casualties managed by a PHTLS® qualified team. We compared outcome between groups. RESULTS: During the study period, 187,839 rescue operations were executed and 280 patients were included. No differences were seen in patient characteristics, trauma severity or geographical distances between groups. Transfer times were significantly reduced in PHTLS® teams than non-qualified teams (9.3 vs. 10.5 min, P = 0.006). Furthermore, the in-field operation times were significantly reduced in PHTLS® qualified teams (36.2 vs. 42.6 min, P = 0.003). Emergency physician involvement did not differ between groups. CONCLUSION: This is the first study to show that the implementation of PHTLS® algorithms in a European metropolitan area is associated with improved efficiency of pre-hospital care for the severely injured. We therefore recommend considering the introduction of PHTLS® in metropolitan areas in the first world.

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells.

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.

Stapes piston insertion depth and clinical correlations.

Background: The insertion of the stapes piston within the vestibule provides the physical basis for a successful stapedotomy. An insertion depth of 0.5 mm is recommended to avoid the dislocation of the stapes prosthesis (e.g. sneezing). Aims: The objective of this study is to analyze the depth of stapes prosthesis insertion and its correlation with clinical outcome. Material and methods: We observed in a retrospective case series 39 otosclerosis patients after a stapedotomy and a postoperative performed flat panel tomography/cone beam CT. The evaluation included the radiologically found depth of prosthesis insertion within the vestibule, the vestibule depth, and the correlation with the bone conduction (BC) threshold, vertigo, and tinnitus. Results: Insertion depth varied between 0.2 and 1.6 mm (mean 0.74 mm). The ratio of insertion depth versus the vestibule depth was between 8% and 59% (mean 26.6%). We observed no correlation between the insertion depth, the length of the prosthesis, the ratio of insertion depth/vestibule depth, postoperative BC, appearance of vertigo, or tinnitus. Conclusions and significance: In our group, we observed no significant relation between insertion depth of the stapes piston, postoperative vertigo, tinnitus, or decrease of the BC.