Neurons are highly polarized cells that depend on mitochondria for energy and signaling homeostasis. Importantly, energy and signaling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyze mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction of individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with targeted mitochondrial gene expression tracking and mtDNA profiling to develop a platform for live-cell single-mitochondrion tracking and analysis. By tracking the expression of specific mitochondrially encoded genes in the same neurons over time, we reveal preliminary data showing a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to α-synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides a proof-of-concept for precise, temporal measurements of mitochondrial composition and targeted gene expression in vitro at single-organelle resolution, opening opportunities for single-cell and single-organelle studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology, and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, and stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein-AM, and nuclear stain Hoechst-33,342. Images are acquired in live cells and analyzed using automated image analysis software.

Emerging techniques for mapping mRNAs within the subcellular compartments of live cells hold great promise for advancing our understanding of the spatial distribution of transcripts and enabling the study of single-cell dynamics in health and disease. This is particularly critical for polarized cells, such as neurons, where mRNA compartmentalization is essential for regulating gene expression, and defects in these localization mechanisms are linked to numerous neurological disorders. However, many subcellular analysis techniques require a compromise between subcellular precision, live-cell measurements, and nondestructive access to single cells in their native microenvironment. To overcome these challenges, we employ a single-cell technology that we have recently developed, the nanotweezer, which features a nanoscale footprint (∼100 nm), avoids cytoplasmic fluid aspiration, and enables rapid RNA isolation from living cells with minimal invasiveness. Using this tool, we investigate single-cell mRNA compartmentalization in the soma and dendrites of hippocampal neurons at different stages of neuronal development. By combining precise targeting with sequential sampling, we track changes in mRNA abundance at dendritic spine regions of the same neuron, both before and after stimulation. This minimally invasive approach enables time-resolved, subcellular gene expression profiling of the same single cell. This could provide critical insights into polarized cells and advance our understanding of biological processes and complex diseases.

Quantitatively assessing the spatial distribution of biomolecules in individual cells can aid our understanding of the roles that they have within a cell and therefore help us elucidate their molecular behaviour under different cellular conditions and in disease. Subcellular extraction methods have the potential to achieve this via multi-omics analyses from single cells, without the need for lysing or fixing whole cells. However, current intracellular analysis tools are limited in their ability to perform repeated spatially controlled measurements of the same cell because they involve the aspiration of cytoplasmic fluid, cellular alterations, or the destruction of the cell. They also have costly and complicated fabrication procedures. The nanotweezer is a tool that circumvents these issues via direct trapping of biomolecules using dielectrophoresis, allowing for minimally invasive extraction of DNA, RNA and single organelles from living cells, whilst utilising a simple fabrication procedure. Here, we show that the nanotweezer can be used to extract nucleic acids and organelles from subcellular compartments of live single neurons with high spatial precision. Repeat sampling of the same neuron is achieved without affecting the cells’ viability, microenvironment, and neuronal activity. Further, we show that single organelle and mRNA analysis from individual biopsies can be achieved, highlighting the prominence of this technique for precise quantification and mapping of biomolecules in the same neuron.

The neuronal ceroid lipofuscinoses (NCLs) are a group of common inherited neurodegenerative disorders of childhood. All forms of NCLs are life-limiting with no curative treatments. Most of the 13 NCL genes encode proteins residing in endolysosomal pathways, such as CLN5, a potential lysosomal enzyme. Two induced pluripotent stem cell lines (hiPSCs) were generated from skin fibroblasts of CLN5 disease patients via non-integrating Sendai virus reprogramming. They demonstrate typical stem cell morphology, express pluripotency markers, exhibit trilineage differentiation potential and also successfully differentiate into neurons. These hiPSCs represent a potential resource to model CLN5 disease in a human context and investigate potential therapies.

How does memory shape our understanding of history?

ABSTRACT Long-term changes in synaptic strength form the basis of learning and memory. These changes rely upon energy-demanding mechanisms, which are regulated by local Ca2+ signalling. Mitochondria are optimised for providing energy and buffering Ca2+. However, our understanding of the role of mitochondria in regulating synaptic plasticity is incomplete. Here, we have used optical and electrophysiological techniques in cultured hippocampal neurons and ex vivo hippocampal slices from mice with haploinsufficiency of the mitochondrial Ca2+ uniporter (MCU+/−) to address whether reducing mitochondrial Ca2+ uptake alters synaptic transmission and plasticity. We found that cultured MCU+/− hippocampal neurons have impaired Ca2+ clearance, and consequently enhanced synaptic vesicle fusion at presynapses occupied by mitochondria. Furthermore, long-term potentiation (LTP) at mossy fibre (MF) synapses, a process which is dependent on presynaptic Ca2+ accumulation, is enhanced in MCU+/− slices. Our results reveal a previously unrecognised role for mitochondria in regulating presynaptic plasticity of a major excitatory pathway involved in learning and memory.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Induced pluripotent stem cells and their derivatives have become an important tool for researching disease mechanisms. It is hoped that they could be used to discover new therapies by providing the most reliable and relevant human in vitro disease models for drug discovery. This review will summarize recent efforts to use stem cell-derived neurons for drug screening. We also explain the current hurdles to using these cells for high-throughput pharmaceutical screening and developments that may help overcome these hurdles. Finally, we critically discuss whether induced pluripotent stem cell-derived neurons will come to fruition as a model that is regularly used to screen for drugs to treat neurological diseases. (C) 2019 Elsevier Inc. All rights reserved.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

Autophagy is the process by which cellular proteins and organelles are degraded and recycled and is essential to the survival of cells. Defective autophagic degradation has been linked to many neurodegenerative diseases and in particular lysosomal storage diseases. Here we describe a high-content assay to detect defects in the autophagy pathway in induced pluripotent stem cell-derived neurons. This assay utilizes immunofluorescence to stain autophagosomes and uses automated image analysis to measure changes in autophagosome levels in response to modulators of autophagy.

Induced pluripotent stem cell (iPSC) derived neurons are an excellent in vitro model of neurological diseases that are often used in early stage drug discovery projects. Thus far, the use of iPSC-derived cells in small molecule drug screening has been limited, and one of the reasons for this has been the challenge of miniaturization of iPSC culture and differentiation in low volume microwell plate formats. Here we describe a method of seeding iPSC-derived neurons into 384-well plates towards the end of the differentiation procedure. This method covers coating the plates with substrates to aid attachment, dissociation of the cells into a single cell suspension, and seeding onto 384-well plates to give an even distribution of neurons. This method facilitates the use of iPSC-derived neurons for high-content imaging, whole-well assays, and small-molecule drug screening.

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional 'snapshot' of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10-20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.

Alpha-synuclein aggregation and mitochondrial dysfunction are central to the pathogenesis of Parkinson's disease (PD). This study investigates the structure specific effects of α-synuclein on mitochondrial function and demonstrates the mechanism by which oligomerisation of the protein results in a toxic gain of function within mitochondria. We have previously shown that monomeric α-synuclein enters mitochondria where it interacts with ATP synthase, and aids its efficiency. Employing a proximity ligation assay, we show an interaction between oligomers and ATP synthase, a proposed key component of the mPTP. We demonstrate that beta sheet rich α-synuclein oligomers uniquely induce opening of the mitochondrial permeability transition pore (mPTP) in whole cells and isolated mitochondria. We report that oligomers, but not monomers, generate reactive oxygen species and mitochondrial membrane lipid peroxidation, and these trigger mPTP opening. This oligomer-induced effects lead to neuronal cell death, which could be abrogated by preincubation of the cells with the mPTP inhibitor Cyclosporin A. We validated our findings in an iPS derived neuronal model with an α-synuclein triplication in which increased levels of aggregated α-synuclein cause early onset PD. In this model we report that oligomers also have a strong interaction with the ATP synthase. Furthermore, we demonstrate a low threshold for mPTP opening on exposure to high laser and calcium. mPTP opening in this model led to cell death which could again be prevented by mPTP inhibition. We were thus able to conclude that the mitochondrial abnormalities seen in the iPS cells with high levels of α-synuclein were likely to be mediated by the beta-sheet oligomeric form of the protein. This study provides evidence of a direct effect of oligomers on mitochondria and is the first to specifically link oligomeric α-synuclein to ATP synthase leading to neuronal death.

α-Synuclein is strongly linked to Parkinson’s disease but the molecular targets for its toxicity are not fully clear. However, many neuronal functions damaged in Parkinson’s disease are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling involves close physical associations between the two organelles that are mediated by binding of the integral ER protein vesicle-associated membrane protein-associated protein B (VAPB) to the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). VAPB and PTPIP51 thus act as a scaffold to tether the two organelles. Here we show that α-synuclein binds to VAPB and that overexpression of wild-type and familial Parkinson’s disease mutant α-synuclein disrupt the VAPB-PTPIP51 tethers to loosen ER–mitochondria associations. This disruption to the VAPB-PTPIP51 tethers is also seen in neurons derived from induced pluripotent stem cells from familial Parkinson’s disease patients harbouring pathogenic triplication of the α-synuclein gene. We also show that the α-synuclein induced loosening of ER–mitochondria contacts is accompanied by disruption to Ca2+ exchange between the two organelles and mitochondrial ATP production. Such disruptions are likely to be particularly damaging to neurons that are heavily dependent on correct Ca2+ signaling and ATP.