Pathogenic tau induces an adaptive elevation in mRNA translation rate at early stages of disease.

Alterations in the rate and accuracy of messenger RNA (mRNA) translation are associated with aging and several neurodegenerative disorders, including Alzheimer's disease and related tauopathies. We previously reported that error-containing RNA that are normally cleared via nonsense-mediated mRNA decay (NMD), a key RNA surveillance mechanism, are translated in the adult brain of a Drosophila model of tauopathy. In the current study, we find that newly-synthesized peptides and translation machinery accumulate within nuclear envelope invaginations that occur as a consequence of tau pathology, and that the rate of mRNA translation is globally elevated in early stages of disease in adult brains of Drosophila models of tauopathy. Polysome profiling from adult heads of tau transgenic Drosophila reveals the preferential translation of specific mRNA that have been previously linked to neurodegeneration. Unexpectedly, we find that panneuronal elevation of NMD further elevates the global translation rate in tau transgenic Drosophila, as does treatment with rapamycin. As NMD activation and rapamycin both suppress tau-induced neurodegeneration, their shared effect on translation suggests that elevated rates of mRNA translation are an early adaptive mechanism to limit neurodegeneration. Our work provides compelling evidence that tau-induced deficits in NMD reshape the tau translatome by increasing translation of RNA that are normally repressed in healthy cells.

m6A-dependent circular RNA formation mediates tau-induced neurotoxicity.

Circular RNAs (circRNAs), covalently closed RNA molecules that form due to back-splicing of RNA transcripts, have recently been implicated in Alzheimer's disease and related tauopathies. circRNAs are regulated by N6-methyladenosine (m6A) RNA methylation, can serve as "sponges" for proteins and RNAs, and can be translated into protein via a cap-independent mechanism. Mechanisms underlying circRNA dysregulation in tauopathies and causal relationships between circRNA and neurodegeneration are currently unknown. In the current study, we aimed to determine whether pathogenic forms of tau drive circRNA dysregulation and whether such dysregulation causally mediates neurodegeneration. We identify circRNAs that are differentially expressed in the brain of a Drosophila model of tauopathy and in induced pluripotent stem cell (iPSC)-derived neurons carrying a tau mutation associated with autosomal dominant tauopathy. We leverage Drosophila to discover that depletion of circular forms of muscleblind (circMbl), a circRNA that is particularly abundant in brains of tau transgenic Drosophila, significantly suppresses tau neurotoxicity, suggesting that tau-induced circMbl elevation is neurotoxic. We detect a general elevation of m6A RNA methylation and circRNA methylation in tau transgenic Drosophila and find that tau-induced m6A methylation is a mechanistic driver of circMbl formation. Interestingly, we find that circRNA and m6A RNA accumulate within nuclear envelope invaginations of tau transgenic Drosophila and in iPSC-derived cerebral organoid models of tauopathy. Taken together, our studies add critical new insight into the mechanisms underlying circRNA dysregulation in tauopathy and identify m6A-modified circRNA as a causal factor contributing to neurodegeneration. These findings add to a growing literature implicating pathogenic forms of tau as drivers of altered RNA metabolism.

Profiling senescent cells in human brains reveals neurons with CDKN2D/p19 and tau neuropathology.

Senescent cells contribute to pathology and dysfunction in animal models1. Their sparse distribution and heterogenous phenotype have presented challenges for detecting them in human tissues. We developed a senescence eigengene approach to identify these rare cells within large, diverse populations of postmortem human brain cells. Eigengenes are useful when no single gene reliably captures a phenotype, like senescence; they also help to reduce noise, which is important in large transcriptomic datasets where subtle signals from low-expressing genes can be lost. Each of our eigengenes detected ~2% senescent cells from a population of ~140,000 single nuclei derived from 76 postmortem human brains with various levels of Alzheimer's disease (AD) pathology. More than 97% of the senescent cells were excitatory neurons and overlapped with tau-containing neurofibrillary tangles (NFTs). Cyclin dependent kinase inhibitor 2D (CDKN2D/p19) was predicted as the most significant contributor to the primary senescence eigengene. RNAscope and immunofluorescence confirmed its elevated expression in AD brain tissue whereby p19-expressing neurons had 1.8-fold larger nuclei and significantly more cells with lipofuscin than p19-negative neurons. These hallmark senescence phenotypes were further elevated in the presence of NFTs. Collectively, CDKN2D/p19-expressing neurons with NFTs represent a unique cellular population in human AD with a senescence phenotype. The eigengenes developed may be useful in future senescence profiling studies as they accurately identified senescent cells in snRNASeq datasets and predicted biomarkers for histological investigation.

Cell Culture and Coculture for Oncological Research in Appropriate Microenvironments.

With the increase in knowledge on the importance of the tumor microenvironment, cell culture models of cancers can be adapted to better recapitulate physiologically relevant situations. Three main microenvironmental factors influence tumor phenotype: the biochemical components that stimulate cells, the fibrous molecules that influence the stiffness of the extracellular matrix, and noncancerous cells like epithelial cells, fibroblasts, endothelial cells, and immune cells. Here we present methods for the culture of carcinomas in the presence of a matrix of specific stiffness, and for the coculture of tumors and fibroblasts as well as epithelial cells in the presence of matrix. Information is provided to help with choice and assessment of the matrix support and in working with serum-free medium. Using the example of a tissue chip recapitulating the environmental geometry of carcinomas, we also highlight the development of engineered platforms that provide exquisite control of cell culture parameters necessary in research and development. © 2019 by John Wiley & Sons, Inc.

Gradient-on-a-Chip with Reactive Oxygen Species Reveals Thresholds in the Nucleus Response of Cancer Cells Depending on the Matrix Environment.

Oxidative stress-mediated cancer progression depends on exposure to reactive oxygen species (ROS) in the extracellular matrix (ECM). To study the impact of ROS levels on preinvasive breast cancer cells as a function of ECM characteristics, we created a gradient-on-a-chip in which H2O2 progressively mixes with the cell culture medium within connected microchannels and diffuses upward into the ECM of the open cell culture window. The device utilizes a paper-based microfluidic bifurcating mixer insert to prevent leakage and favor an even fluid distribution. The gradient was confirmed by measuring H2O2 catalyzed into oxygen, and increasing oxidative DNA damage and protective (AOP2) response were recorded in 2D and ECM-based 3D cell cultures. Interestingly, the impact of ROS on nuclear shape and size (annunciating phenotypical changes) was governed by the stiffness of the collagen I matrix, suggesting the existence of thresholds for the phenotypic response to microenvironmental chemical exposure depending on ECM conditions.

Mining the epigenetic landscape of tissue polarity in search of new targets for cancer therapy.

The epigenetic nature of cancer encourages the development of inhibitors of epigenetic pathways. Yet, the clinical use for solid tumors of approved epigenetic drugs is meager. We argue that this situation might improve upon understanding the coinfluence between epigenetic pathways and tissue architecture. We present emerging information on the epigenetic control of the polarity axis, a central feature of epithelial architecture created by the orderly distribution of multiprotein complexes at cell-cell and cell-extracellular matrix contacts and altered upon cancer onset (with apical polarity loss), invasive progression (with basolateral polarity loss) and metastatic development (with basoapical polarity imbalance). This information combined with the impact of polarity-related proteins on epigenetic mechanisms of cancer enables us to envision how to guide the choice of drugs specific for distinct epigenetic modifiers, in order to halt cancer development and counter the consequences of polarity alterations.