MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

Inhibition of GCK-IV kinases dissociates cell death and axon regeneration in CNS neurons.

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.