A Single Human Neuron Approach to Synapse Function

A Single Human Neuron Approach to Synapse Function

Trends in Molecular Medicine Spotlight A Single Human Neuron Approach to Synapse Function Pascal Fenske1,2 and Christian Rosenmund 1,2, * To unde...

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Trends in Molecular Medicine

Spotlight

A Single Human Neuron Approach to Synapse Function Pascal Fenske1,2 and Christian Rosenmund

1,2,

*

To understand human neuronal function, it is crucial to obtain knowledge of how human synapses operate. New approaches are necessary to define the unique properties of human synapses. Recently, new culturing approaches have been developed to obtain cultures of single human neurons for the first time (Rhee et al., Cell Rep. 2019, Meijer et al., Cell Rep. 2019, and Fenske et al., Sci. Rep. 2019). Advances in the analysis of the human genome led to the discovery of many new monogenetic disorders of the central nervous system (CNS). While this has benefitted the diagnosis of rare diseases, understanding the underlying pathophysiology is a greater challenge. So far, studies of model organisms, such as Caenorhabditis elegans, Drosophila, fish, and rodents, have shown some success in recapitulating both organismal and cellular aspects of genetic defects [1]. Recent technological advances in human stem cell research now provide access to human neurons through the establishment of induced pluripotent stem cells (iPSCs) and neuronal differentiation protocols [2–4]. Two potential advantages of cultured human neurons are that they better reflect the human biology compared with their animal counterparts and that they are amendable for studying complex genetic diseases when iPSCs are derived from patients.

This holds true in particular for the maturation of synaptic transmission, indicative of neuronal maturity and arguably one of the most critical neuronal functions. While mouse neuron synapses mature within 2 weeks in vitro, human neurons take 6 weeks or more. Therefore, only masscultured human neurons have been utilized to study human synapse function [3,4], where the depth of analysis and quantification of synaptic properties and parameters is limited due to the complex network formed. A better alternative is autaptic cultures, in which single neurons grow in isolation on astrocytic microislands and form synapses exclusively with themselves. They provide better analytical power for the differential analysis of human synaptic dysfunctions; the simple connectivity patterns allow for precise characterization of synaptic input and output [6] simultaneously at the level of both morphology and function [7]. However, autaptic cultures have a limited experimental life time, with deteriorations of neuronal health after 3–4 weeks in culture. Therefore, autaptic recordings have, up until now, only been available for rodent neurons.

Three research groups have tackled this road block and recently published protocols that increase the feasibility of a single human neuron-culturing approach. Studies by Rhee et al. [8] and Meijer et al. [9] focused on improving the in vitro life time of single-neuron cultures. Testing different media composition and astrocyte origins (mouse, rat, and human), both groups developed an optimized protocol with mouse astrocytes and media supplemented with a small amount of fetal calf serum. These modifications led to gradual but significant improvements, enabling human neurons to live long enough to acquire mature synapses. However, in both studies, cultivation times of 5–12 weeks were still necHowever, a disadvantage is that cultured essary to obtain reliable physiological human neurons differentiate more slowly parameters for synaptic transmission, limwhen compared with mouse neurons [5]. iting the yield of successful recordings.

Fenske et al. [10] used a different approach to obtain mature human neurons for autaptic cultures. Rather than trying to extend the life time of human singlecultured neurons, they allowed the maturation of human neurons to proceed in mass culture, and subsequently transferred already-mature neurons onto microislands by replating. That this approach was successful was surprising, given the observation that mature rodent neurons cannot be replated due to the massive cell death that occurs after replating. Interestingly, human neurons appear more robust, surviving the transfer even after 3 months in mass culture and being resilient to damage during replating. The neurons consistently regrew functional synapses within 2 weeks, allowing for detailed functional and morphological analyses with high yield. In addition to these significant improvements in feasibility, the results from the autaptic culture experiments demonstrated new insights into human neuronal functions and provide a promising approach to enable us to better analyze human neuronal dysfunction in the future. For example, the studies demonstrated precise measurements of synapse numbers formed by glutamatergic neurons [8–10], and also how efficient action potentials are transduced into synaptic vesicle fusion [8,10]. Thus, dysfunction in synapse formation or impaired reliability of synaptic transmission are more easily detected. The studies also addressed putative cell line-dependent variability in neuronal properties; all studies used several iPSC lines as independent replicates and synapses derived from different healthy iPSCs showed only minor significant differences between different donor cells. Future studies will need to determine whether these differences are only intrinsic, underscoring the need for isogenic controls, or whether these differences also depend on the protocols used. Trends in Molecular Medicine, July 2019, Vol. 25, No. 7

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The three studies further demonstrated modulation of neurotransmitter release by GABAB/mGluR G-protein-coupled receptors and phorbol esters [8,10], and complemented the experimental portfolio by including induced GABAergic neurons in the analysis [8,9]. They also demonstrated the first full assessment of synaptic properties from genetically modified glutamatergic neurons recapitulating critical functions of the vesicle-priming factor Munc13-1 at the human synapse [10].

genomic quality control checks for SNPs and copy number variation after the neuronal differentiation of iPSCs [9] will help to improve the reliability of such experiments. The morphological and functional properties of autaptic neurons and synapses are accessible to highly quantitative analysis, which may standardize future analyses of drug- or genetically induced phenotypes with higher precision as well as opening more sophisticated analyses methods than is possible with current culture methods. The protocols are also scalable by cryopreservation of alreadyinduced neurons [8,9]. Thus, defined batches of different subtypes of neurons provide a high level of standardization.

Overall, these findings were relatively consistent with data obtained from rodent models. The progress presented in these three papers is promising, and further protocol improvements, possibly by combining all three approaches (Figure 1), will likely further improve the yield and quality of single human neurons for physi- Although the neurons reported here are ological experiments. In addition, stringent single cultured neurons and, therefore,

amendable mainly for the analysis of cell autonomous functions and dysfunctions, the presented approaches could allow the systematic analysis of fundamental human neuron functions, morphologies, and gene expression. Such approaches will also benefit from modified induction protocols that recapitulate the diversity of neuronal cell types displayed in vivo. We believe that single-neuron physiology analyses are also reliable methods for identifying synaptic phenotypes caused by mutations and will pave the way for deciphering the underlying pathophysiology of patients with neurological and neuropsychiatric disorders of genetic origin. The integration of CRISPR-edited lines with isogenic controls into more complex models of human neurons may enable the targeted investigation of specific mutations and their impact on synaptic transmission.

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Figure 1. Schematic Diagram for the Generation of Human Autaptic Neurons. Donor cells are isolated from patient samples and used for the generation of induced pluripotent stem cells (iPSCs). After induction of different neuronal subtypes, induced neurons can be either cryopreserved, plated directly on astrocytic microislands for maturation, or kept in mass culture followed by dissociation and replating steps. Astrocytes for co-culture and the generation of microislands are obtained from rodent brains.

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Institute of Neurophysiology, Charité – Universitätsmedizin, 10117, Berlin, Germany 2 NeuroCure Cluster of Excellence, Charité – Universitätsmedizin, 10117, Berlin, Germany 1

*Correspondence: [email protected] (C. Rosenmund).

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https://doi.org/10.1016/j.molmed.2019.05.005 © 2019 Elsevier Ltd. All rights reserved.

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Sampathkumar, C. et al. (2016) Loss of MeCP2 disrupts cell autonomous and autocrine BDNF signaling in mouse glutamatergic neurons. eLife 5, e19374 8. Rhee, H.J. et al. (2019) An autaptic culture system for standardized analyses of iPSC-derived human neurons. Cell Rep. 27, 2212–2228 9. Meijer, M. et al. (2019) A single-cell model for synaptic transmission and plasticity in human iPSC-derived neurons. Cell Rep. 27, 2199–2211 10. Fenske, P. et al. (2019) Autaptic cultures of human induced neurons as a versatile platform for studying synaptic function and neuronal morphology. Sci. Rep. 9, 4890

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