Sonlicromanol improves neuronal network dysfunction and transcriptome changes linked to m.3243A>G heteroplasmy in iPSC-derived neurons.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.

Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.

Immuno-detection by sequencing enables large-scale high-dimensional phenotyping in cells.

Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We use ID-seq to simultaneously measure 70 (phospho-)proteins in primary human epidermal stem cells to screen the effects of ~300 kinase inhibitor probes to characterise the role of 225 kinases. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations.

A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.