Biology subjects

Kampmann, M.

Publications and source records attributed to Kampmann, M..

60 records · Page 4Linked to original sources

bioRxiv2019

Mitochondrial dysfunction is signaled to the integrated stress response by OMA1, DELE1 and HRI

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2 phosphorylation upregulates the transcription factor ATF4. However, how mitochondrial stress is relayed to the ISR is unknown. We found that HRI is the eIF2 kinase necessary and sufficient for this relay. Using an unbiased CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a little-characterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates the OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2 kinase activity. Blockade of the OMA1-DELE1-HRI pathway is beneficial during some, but not all types of mitochondrial stress, and leads to an alternative response that induces specific molecular chaperones. Therefore, this pathway is a potential therapeutic target enabling fine-tuning of the ISR for beneficial outcomes in diseases involving mitochondrial dysfunction.

cell biology↗

bioRxiv2019

Compromised function of an ESCRT complex promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimers Disease and frontotemporal dementia. However, we lack a systematic understanding of the cellular path-ways controlling prion-like propagation. To uncover such pathways, we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation. Our screens uncovered that knockdown of several components of the ESCRT machinery, including CHMP6, or CHMP2A in combination with CHMP2B (a gene linked to familial fronto-temporal dementia), promote propagation of tau aggregation. We found that knockdown of these genes caused damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation.

cell biology↗

bioRxiv2019

CRISPR-based platform for multimodal genetic screens in human iPSC-derived neurons

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

cell biology↗

bioRxiv2017

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs)

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimers disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tauheparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse organotypic brain slice culture. Together, these results suggest novel strategies to halt tau transmission.

neuroscience↗

bioRxiv2017

Decoding directional genetic dependencies through orthogonal CRISPR/Cas screens

Genetic interaction studies are a powerful approach to identify functional interactions between genes. This approach can reveal networks of regulatory hubs and connect uncharacterised genes to well-studied pathways. However, this approach has previously been limited to simple gene inactivation studies. Here, we present an orthogonal CRISPR/Cas-mediated genetic interaction approach that allows the systematic activation of one gene while simultaneously knocking out a second gene in the same cell. We have developed this concept into a quantitative and scalable combinatorial screening platform that allows the parallel interrogation of hundreds of thousands of genetic interactions. We demonstrate that the established platform works robustly to uncover genetic interactions in human cancer cells and to interpret the direction of the flow of genetic information.

molecular biology↗

bioRxiv2017

CRISPulator: A Discrete Simulation Tool For Pooled Genetic Screens

The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on optimal screen design, which also affects cost and scalability. We present CRISPulator, a computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy. We illustrate its power by deriving non-obvious rules for optimal screen design.

bioinformatics↗