Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. ...Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the
G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in
G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing
G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.
Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by ...posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo.
•Caspase-2 cleaves Tau at D65 and D421.•In vivo overexpression leads to low steady-state protein levels for caspase-2 generated Tau fragments.•CHIP specifically co-immunoprecipitates with Tau fragments in vivo.•Caspase-2 generated Tau fragments are preferentially ubiquitinated by CHIP.•CHIP is linked to accelerated degradation of caspase-2 generated Tau fragments in vitro.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has ...been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments.
•Generation of isogenic WT and P525L FUS-eGFP reporter iPSCs•P525L FUS-eGFP SGs are more numerous, more intense, and larger than WT•Increasing PI3K/AKT/mTOR-regulated autophagy reduces FUS-eGFP recruitment to SGs•Brain-penetrant drugs that induce autophagy ameliorate the FUS SG phenotype
Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG) phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immune-related adverse events (irAEs) are a class-effect of checkpoint inhibitors (CIs). The development of a Bullous pemphigoid (BP)-like blistering disease, driven by autoantibodies against the ...hemidesmosomal protein BP180, is a potentially serious irAE whose incidence seems to be increasing. We therefore set out to characterize the clinical and (immuno)histopathological features and treatment responses of cases of BP which developed during or after CI therapy collated in six German tertiary referral centers between 2014 and 2018. We identified twelve cases of BP which emerged during and/or after CI therapy. The time interval between the initiation of CI therapy and the diagnosis of BP was 3-74 weeks (median: 23 weeks). Age at the time of diagnosis of BP varied between 62 and 80 years (median: 76 years). The clinical presentation of the patients was diverse but the severity was relatively mild when compared to that seen in most cases of spontaneous BP. Only four patients met all of the immunopathological criteria recommended in the European guidelines for the diagnosis of BP. Topical corticosteroid treatment was sufficient to achieve disease control in most patients. CI therapy could be continued in 8 out of 12 patients. In summary, our study indicates that cases of BP during or after CI therapy bear several peculiarities distinguishing them from spontaneous BP. Given the diversity of the clinical presentation of CI-induced BP the application of existing diagnostic algorithms developed for spontaneous BP can be utilized to uncover the frequency and features of CI-induced BP and to develop and optimize management algorithms.
Induced pluripotent stem cells (iPSCs) have recapitulated several aspects of Parkinson's disease (PD), but most iPSCs are derived from familial cases, which account for only about 15% of patients. ...Thus, while the emphasis has justifiably been on using iPSCs to model rare familial cases, models for the most common forms of PD are critically lacking. Here, we report the generation of an iPSC-based model of idiopathic PD (iPD) with or without RS1491923, which is a common risk variant in the LRRK2 locus. Consistent with GWA studies, we found large variability in our datasets. However, iPSC-derived neurons carrying the risk allele emerged for displaying subtle disturbances of cellular degradative systems, in line with familial PD models. We also observed that treatment with the LRRK2 inhibitor CZC-25146 slightly reduced a marker of aSYN pathology in all iPD lines. Future iPSC-based studies may need to be structured similarly to large GWA studies in order to obtain relevant statistical power. However, results from this pilot study suggest that iPSC-based modeling represents an attractive way to investigate idiopathic diseases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BackgroundUveal melanoma (UM) is highly refractory to treatment with dismal prognosis in advanced stages. The value of the combined checkpoint blockade with CTLA-4 and PD-1 inhibition in metastatic ...UM is currently unclear.MethodsPatients with metastatic or unresectable UM treated with ipilimumab in combination with a PD-1 inhibitor were collected from 16 German skin cancer centers. Patient records of 64 cases were analyzed for response, progression-free survival (PFS), overall survival (OS), and safety. Clinical parameters and serum biomarkers associated with OS and treatment response were determined with Cox regression modelling and logistic regression.ResultsThe best overall response rate to combined checkpoint blockade was 15.6% with 3.1 and 12.5% complete and partial response, respectively. The median duration of response was 25.5 months (range 9.0–65.0). Stable disease was achieved in 21.9%, resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0–65.0). The median PFS was 3.0 months (95% CI 2.4–3.6). The median OS was estimated to 16.1 months (95% CI 12.9–19.3). Regarding safety, 39.1% of treated patients experienced a severe, treatment-related adverse event according to the CTCAE criteria (grade 3: 37.5%; grade 4: 1.6%). The most common toxicities were colitis (20.3%), hepatitis (20.3%), thyreoiditis (15.6%), and hypophysitis (7.8%). A poor ECOG performance status was an independent risk factor for decreased OS (p = 0.007).ConclusionsThe tolerability of the combined checkpoint blockade in UM may possibly be better than in trials on cutaneous melanoma. This study implies that combined checkpoint blockade represents the hitherto most effective treatment option available for metastatic UM available outside of clinical trials.
Patients with skin cancer increasingly watch online videos to acquire disease-related information. Until now, no scientific evaluation of the quality of videos available for German-speaking patients ...with basal cell carcinoma (BCC) has been performed.
In this study, we aimed to identify and evaluate videos about BCC provided on YouTube.
A video search on YouTube was conducted in July 2020, using German BCC-related keywords (eg, "Basalzellkarzinom," "Basaliom," "weißer hautkrebs," and "heller hautkrebs"). The first three pages (ie, 60 videos) were searched by two independent researchers for each keyword. Two authors evaluated videos that met the predefined eligibility criteria. The quality of the information of the videos was evaluated using the DISCERN tool and the Global Quality Scale (GQS). The understandability and actionability were assessed with the Patient Education Materials Assessment Tool for Audiovisual Materials (PEMAT-A/V). The reliability was assessed with the JAMA (Journal of the American Medical Association) criteria score. Subgroup differences were identified using the Kruskal-Wallis test.
A total of 41 videos were included in the evaluation. The mean assessment scores were as follows: DISCERN, 3.3 (SD 0.80); GQS, 3.8 (SD 1.1); JAMA, 27.74% (SD 22.1%); understandability, 70.8% (SD 13.3%); and actionability, 45.9% (SD 43.7%). These values indicated that the videos were of medium to good quality and had good understandability, low actionability, and poor reliability. The quality of videos provided by health professionals was significantly higher than that of videos provided by laypersons.
Optimization of health-related videos about BCC is desirable. In particular, adaptation to reliability criteria is necessary to support patient education and increase transparency.
Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified ...small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3β is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3β. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3β in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.
•Phenotypic screening identifies CDK inhibitors protecting neurons from inflammation•Inhibition of CDK5 is involved in neuroprotection but is not sufficient•Dual inhibition of CDK5 and GSK3β is neuroprotective in vitro and in vivo•Quantitative phosphoproteomics links neuroprotection to microtubule dynamics
Neuroinflammation is a hallmark of many neurological disorders and can be neurotoxic. Sterneckert and colleagues perform a phenotypic screening campaign identifying compounds protecting motor neurons from inflammation. Compound profiling demonstrates that dual inhibition of CDK5 and GSK3β mediates neuroprotection. Quantitative phosphoproteomics demonstrates that neuroprotection is linked to alterations in microtubule dynamics mediated by phosphorylation of microtubule-associated proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Skin cancer is one of the most common cancers worldwide and the number of patients is steadily increasing. In skin cancer care, greater interdisciplinary cooperation is required for prevention, early ...detection, and new complex systemic therapies. However, the implementation of innovative medical care is a major challenge, especially for rural regions with an older than average, multimorbid population, with limited mobility, that are long distances from medical facilities. Solutions are necessary to ensure comprehensive oncological care in rural regions. The aim of this study was to identify indicators to establish a regional care network for integrated skin cancer care. To capture the perspectives of different stakeholder groups, we conducted two focus groups with twenty skin cancer patients and their relatives, a workshop with eight physicians, and three semi-structured interviews with health insurance company representatives. Qualitative data were recorded, transcribed, and analyzed following Mayring's content analysis methods. We generated ten categories based on the reported optimization potentials; five categories were assigned to all three stakeholder groups: Prevention and early diagnosis, accessibility of physicians/clinics, physicians' resources, care provider's responsibilities, and information exchange. The results indicate the need for stronger integration of care in the region. They provide the basis for regional networking as, for example, the conception of treatment pathways or telemedicine with the aim to improve a comprehensive skin cancer care. Our study should raise awareness and postulate as a demand that all patients receive guideline-based therapy, regardless of where they live.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK, VSZLJ
Adjuvant treatment of melanoma patients with immune-checkpoint inhibition (ICI) and targeted therapy (TT) significantly improved recurrence-free survival. This study investigates the real-world ...situation of 904 patients from 13 German skin cancer centers with an indication for adjuvant treatment since the approval of adjuvant ICI and TT. From adjusted log-binomial regression models, we estimated relative risks for associations between various influence factors and treatment decisions (adjuvant therapy yes/no, TT vs. ICI in
mutant patients). Of these patients, 76.9% (95% CI 74-80) opted for a systemic adjuvant treatment. The probability of starting an adjuvant treatment was 26% lower in patients >65 years (RR 0.74, 95% CI 68-80). The most common reasons against adjuvant treatment given by patients were age (29.4%, 95% CI 24-38), and fear of adverse events (21.1%, 95% CI 16-28) and impaired quality of life (11.9%, 95% CI 7-16). Of all
-mutated patients who opted for adjuvant treatment, 52.9% (95% CI 47-59) decided for ICI. Treatment decision for TT or ICI was barely associated with age, gender and tumor stage, but with comorbidities and affiliated center. Shortly after their approval, adjuvant treatments have been well accepted by physicians and patients. Age plays a decisive role in the decision for adjuvant treatment, while pre-existing autoimmune disease and regional differences influence the choice between TT or ICI.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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