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Background: PINK1 deficiency causes the autosomal recessive PARK6 variant of Parkinson’s disease. PINK1 activates ubiquitin by phosphorylation and cooperates with the downstream ubiquitin ligase PARKIN, to exert quality control and control autophagic degradation of mitochondria and of misfolded proteins in all cell types.
Methods: Global transcriptome profiling of mouse brain and neuron cultures were assessed in protein-protein interaction diagrams and by pathway enrichment algorithms. Validation by quantitative reverse transcriptase polymerase chain reaction and immunoblots was performed, including human neuroblastoma cells and patient primary skin fibroblasts.
Results: In a first approach, we documented Pink1-deleted mice across the lifespan regarding brain mRNAs. The expression changes were always subtle, consistently affecting “intracellular membrane-bounded organelles”. Significant anomalies involved about 250 factors at age 6 weeks, 1300 at 6 months, and more than 3500 at age 18 months in the cerebellar tissue, including Srsf10, Ube3a, Mapk8, Creb3, and Nfkbia. Initially, mildly significant pathway enrichment for the spliceosome was apparent. Later, highly significant networks of ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing occurred. Finally, an enrichment of neuroinflammation factors appeared, together with profiles of bacterial invasion and MAPK signaling changes—while mitophagy had minor significance. Immunohistochemistry showed pronounced cellular response of Iba1-positive microglia and GFAP-positive astrocytes; brain lipidomics observed increases of ceramides as neuroinflammatory signs at old age.
In a second approach, we assessed PINK1 deficiency in the presence of a stressor. Marked dysregulations of microbial defense factors Ifit3 and Rsad2 were consistently observed upon five analyses: (1) Pink1 −/− primary neurons in the first weeks after brain dissociation, (2) aged Pink1 −/− midbrain with transgenic A53T-alpha-synuclein overexpression, (3) human neuroblastoma cells with PINK1-knockdown and murine Pink1 −/− embryonal fibroblasts undergoing acute starvation, (4) triggering mitophagy in these cells with trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and (5) subjecting them to pathogenic RNA-analogue poly(I:C). The stress regulation of MAVS, RSAD2, DDX58, IFIT3, IFIT1, and LRRK2 was PINK1 dependent. Dysregulation of some innate immunity genes was also found in skin fibroblast cells from PARK6 patients.
Conclusions: Thus, an individual biomarker with expression correlating to progression was not identified. Instead, more advanced disease stages involved additional pathways. Hence, our results identify PINK1 deficiency as an early modulator of innate immunity in neurons, which precedes late stages of neuroinflammation during alpha-synuclein spreading.
Simple Summary: Therapeutic antibodies are an integral part of treatment regimens for metastasized colorectal cancer. In KRAS wildtype tumors both bevacizumab and cetuximab are active. While bevacizumab has previously been shown to induce tumor hypoxia, we here report that EGFR inhibition by cetuximab protects colon cancer cells from hypoxia-induced cell death. This effect appears to be responsible for the inferior efficacy of a treatment sequence of bevacizumab followed by cetuximab versus an inverse sequence that we observed in a colorectal cancer mouse model. It also offers a mechanistic explanation for effects observed in clinical trials such as underadditive or even detrimental effects when combining bevacizumab and cetuximab (CAIRO2 trial) and the superior efficacy of first line cetuximab (FIRE-3 trial) under chemotherapy backbones in colorectal cancer.
Abstract: Monoclonal antibodies like cetuximab, targeting the epidermal growth factor receptor (EGFR), and bevacizumab, targeting the vascular endothelial growth factor (VEGF), are an integral part of treatment regimens for metastasized colorectal cancer. However, inhibition of the EGFR has been shown to protect human glioma cells from cell death under hypoxic conditions. In colon carcinoma cells, the consequences of EGFR blockade in hypoxia (e.g., induced by bevacizumab) have not been evaluated yet. LIM1215 and SW948 colon carcinoma and LNT-229 glioblastoma cells were treated with cetuximab, PD153035, and erlotinib and analyzed for cell density and viability. The sequential administration of either cetuximab followed by bevacizumab (CET->BEV) or bevacizumab followed by cetuximab (BEV->CET) was investigated in a LIM1215 (KRAS wildtype) and SW948 (KRAS mutant) xenograft mouse model. In vitro, cetuximab protected from hypoxia. In the LIM1215 model, a survival benefit with cetuximab and bevacizumab monotherapy was observed, but only the sequence CET->BEV showed an additional benefit. This effect was confirmed in the SW948 model. Our observations support the hypothesis that bevacizumab modulates the tumor microenvironment (e.g., by inducing hypoxia) where cetuximab could trigger protective effects when administered later on. The sequence CET->BEV therefore seems to be superior as possible mutual adverse effects are bypassed.
Glioblastoma multiforme (GBM) is treated by surgical resection followed by radiochemotherapy. Bevacizumab is commonly deployed for anti‐angiogenic therapy of recurrent GBM; however, innate immune cells have been identified as instigators of resistance to bevacizumab treatment. We identified angiopoietin‐2 (Ang‐2) as a potential target in both naive and bevacizumab‐treated glioblastoma. Ang‐2 expression was absent in normal human brain endothelium, while the highest Ang‐2 levels were observed in bevacizumab‐treated GBM. In a murine GBM model, VEGF blockade resulted in endothelial upregulation of Ang‐2, whereas the combined inhibition of VEGF and Ang‐2 leads to extended survival, decreased vascular permeability, depletion of tumor‐associated macrophages, improved pericyte coverage, and increased numbers of intratumoral T lymphocytes. CD206+ (M2‐like) macrophages were identified as potential novel targets following anti‐angiogenic therapy. Our findings imply a novel role for endothelial cells in therapy resistance and identify endothelial cell/myeloid cell crosstalk mediated by Ang‐2 as a potential resistance mechanism. Therefore, combining VEGF blockade with inhibition of Ang‐2 may potentially overcome resistance to bevacizumab therapy.
Downstream effects of plectin mutations in epidermolysis bullosa simplex with muscular dystrophy
(2016)
Mutations of the human plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In the present study we analyzed the downstream effects of PLEC mutations on plectin protein expression and localization, the structure of the extrasarcomeric desmin cytoskeleton, protein aggregate formation and mitochondrial distribution in skeletal muscle tissue from three EBS-MD patients.
PLEC gene analysis in a not previously reported 35-year-old EBS-MD patient with additional disease features of cardiomyopathy and malignant arrhythmias revealed novel compound heterozygous (p.(Phe755del) and p.(Lys1040Argfs*139)) mutations resulting in complete abolition of plectin protein expression. In contrast, the other two patients with different homozygous PLEC mutations showed preserved plectin protein expression with one only expressing rodless plectin variants, and the other markedly reduced protein levels. Analysis of skeletal muscle tissue from all three patients revealed severe disruption of the extrasarcomeric intermediate filament cytoskeleton, protein aggregates positive for desmin, syncoilin, and synemin, degenerative myofibrillar changes, and mitochondrial abnormalities comprising respiratory chain dysfunction and an altered organelle distribution and amount.
Our study demonstrates that EBS-MD causing PLEC mutations universally result in a desmin protein aggregate myopathy phenotype despite marked differences in individual plectin protein expression patterns. Since plectin is the key cytolinker protein that regulates the structural and functional organization of desmin filaments, the defective anchorage and spacing of assembled desmin filaments is the key pathogenetic event that triggers the formation of desmin protein aggregates as well as secondary mitochondrial pathology.
Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs.
Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.
Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.
Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.
Carboxypeptidase E (CPE) has recently been described as a multifunctional protein that regulates proliferation, migration and survival in several tumor entities. In glioblastoma (GBM), the most malignant primary brain tumor, secreted CPE (sCPE) was shown to modulate tumor cell migration. In our current study, we aimed at clarifying the underlying molecular mechanisms regulating anti-migratory as well as novel metabolic effects of sCPE in GBM. Here we show that sCPE activates mTORC1 signaling in glioma cells detectable by phosphorylation of its downstream target RPS6. Additionally, sCPE diminishes glioma cell migration associated with a negative regulation of Rac1 signaling via RPS6, since both inhibition of mTOR and stimulation of Rac1 results in a reversed effect of sCPE on migration. Knockdown of CPE leads to a decrease of active RPS6 associated with increased GBM cell motility. Apart from this, we show that sCPE enhances glucose flux into the tricarboxylic acid cycle at the expense of lactate production, thereby decreasing aerobic glycolysis, which might as well contribute to a less invasive behavior of tumor cells. Our data contributes to a better understanding of the complexity of GBM cell migration and sheds new light on how tumor cell invasion and metabolic plasticity are interconnected.
We provide in this paper a comprehensive comparison of various transfer learning strategies and deep learning architectures for computer-aided classification of adult-type diffuse gliomas. We evaluate the generalizability of out-of-domain ImageNet representations for a target domain of histopathological images, and study the impact of in-domain adaptation using self-supervised and multi-task learning approaches for pretraining the models using the medium-to-large scale datasets of histopathological images. A semi-supervised learning approach is furthermore proposed, where the fine-tuned models are utilized to predict the labels of unannotated regions of the whole slide images (WSI). The models are subsequently retrained using the ground-truth labels and weak labels determined in the previous step, providing superior performance in comparison to standard in-domain transfer learning with balanced accuracy of 96.91% and F1-score 97.07%, and minimizing the pathologist's efforts for annotation. Finally, we provide a visualization tool working at WSI level which generates heatmaps that highlight tumor areas; thus, providing insights to pathologists concerning the most informative parts of the WSI.