Neuro293: A REST-knockout HEK-293 cell line enables the expression of neuron-restricted genes for the high-throughput testing of human neurobiology and the biochemistry of neuronal proteins

Abstract

Efficient interrogation of neurobiology remains bottlenecked by obtaining mature neurons. Immortalized cell lines still require lengthy differentiation periods to obtain neuron-like cells, which may not efficiently differentiate and are challenging to transfect with plasmids relative to other cell lines such as HEK-293's. To overcome challenges with limited access to cells that express mature neuronal proteins, we knocked out the RE1-silencing transcription factor (REST) from HEK-293's to create a novel neuron-like cell, which we name Neuro293. RNA-sequencing and bioinformatics analyses revealed a significant upregulation of genes associated with neurobiology and membrane excitability including pre-/post-synaptic proteins, voltage gated ion channels, neuron-cytoskeleton, as well as neurotransmitter synthesis, packaging, and release. Western blot validated the upregulation of Synapsin-1 (Syn1) and Snap-25 as two neuron-restricted proteins, as well as the potassium channel Kv1.2. Immunocytochemistry against Neurofilament 200 kd revealed a significant upregulation and accumulation in singular processes extending from Neuro293's cell body. Similarly, while Syn1 increased in the cell body, Syn1 protein accumulated at the ends of processes extruding from Neuro293's. Neuro293's express reporter-genes through the Syn1 promoter after infection with adeno-associated viruses (AAV). However, transient transfection with AAV2 plasmids led to leaky expression through promoter-independent mechanisms. Despite an upregulation of many voltage-gated ion channels, Neuro293's do not possess excitable membranes. Collectively, REST-knockout in HEK-293's induces a quickly dividing and easily transfectable cell line that expresses neuron-restricted and mature neuronal proteins which can be used for high-throughput biochemical interrogation, however, without further modifications neither HEK-293's or Neuro293's exhibit properties of excitable membranes.

Keywords: high-throughput testing; mature neuronal proteins; neuronal differentiation; stable cell line.

Figure 1.

Establishing Neuro293’s using CRISPR/Cas9 to knockout REST upregulates neuronal-related gene transcription. CRISPR/Cas9 was used to knockout REST from HEK-239’s, and a single cell was isolated and expanded to establish Neuro293’s (A). PCR and western blot were performed on a single expanded clone (PCR = black and white bands; western blot = colored bands) (A). GO, KEGG, and Reactome enrichment analysis implicated a vast network of neuronal-related genes as upregulated following REST-KO (B) including genes associated with axon development, ion channels, and the regulation of the membrane potential, as well as pre-/post-synaptic functions, including neurotransmitter synthesis and release (C). All genes displayed in the Top 50 DEGS were significant discoveries after FDR correction. See Supplementary Materials 3 for high-resolution Dot Plots from (B). This figure was created with Biorender.com.

Figure 2.

REST-KO stably upregulates neuron-restricted proteins, enabling the high-throughput biochemical interrogation of human neuronal proteins. Neuronal-specific genes Syn1 and Snap25, as well as the non-neuronal-specific ion channel Kv1.2, were upregulated at the protein level using western blot (A). Sub-cellular localization of Syn1 and NFH identifies what appears to be assembly and accumulation along a preferred cellular extension (B and C). Protein accumulation within Neuro293s and HEK-293s was closely predicted by RNA levels identified in RNA-sequencing (B, E, D–G). NeuN was already expressed in HEK-293s while TUBB3 displayed trends towards an increase that were not significant at either the protein or mRNA level. n = 3 replicates/group (A), n = 4–6 images/group (D and F). Scale Bars = 100 µm. Error Bars = SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.

Figure 3.

Transient transfection with plasmids enables protein expression in a promotor-independent manner through AAV2 plasmids, but only Neuro293’s express through the Syn1 promoter after viral transduction. Neuron-restricted promoters Syn1, CamKIIa, and HB9 were tested for the ability to express downstream proteins after transient transfection with AAV2 plasmids (A). Surprisingly, all promoters exhibited reporter-gene expression in both Neuro293 and HEK-293 cells, even when RNA-sequencing indicated an absence of RNA-transcriptions (CamKIIa was not expressed in either cell, and Syn1 was lowly expressed in HEK-293’s; Supplementary Table 3). Paradoxically, reporter gene expression was less active in Neuro293’s for currently unknown reasons, despite a higher level of Syn1 mRNA and protein expression (Figs 1 and 2). Use of packaged AAV vectors to transduce cells, in contrast, demonstrates an expected higher expression of reporter genes through the Syn1 promoter (B). n = 6–8 images/group. Scale Bars = 100 µm. Error Bars = SEM. ****P < 0.0001, ns = not significant.

Figure 4.

REST-KO does not augment membrane excitability despite an upregulation of voltage-gated ion channels. Patch clamp on HEK-293’s and Neuro293’s was used to test membrane excitability through an ascending range of voltage (A and B). Representative family of current traces, Vtest ranging from −80 to +40 in 10-mV increments, with voltage protocol schematic shown above (A). Current/voltage curve fails to show differences in current density between REST-KO and HEK-293’s (B). n = 5 HEK-293 and n = 5 Neuro293 cells/group. Error Bars = SEM.