Mechanisms of inherited neurodegenerative diseases

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Identification of a new mechanism of axonal RNA granule transport: Neurons are highly polarized and compartmentalized cells, with complex dendritic networks and far-reaching axons. To effectively and efficiently respond to stimuli and metabolic demands far from the soma, neurons rely heavily on local translation of proteins. Within axons, RNAs are locally translated at a number of sites, including growth cones, pre-synapses, and at intra-axonal organelles such as mitochondria. Though we know that axonal RNA transport requires microtubules and motor proteins, the precise mechanisms by which RNAs are transported from the soma to these distal regions of the neuron had been unclear. Unlike membrane-bound cargos, such as mitochondria and endosomes, RNAs usually exist within membraneless phase-separated ribonuclear protein complexes known as RNA granules. Therefore, the adapter machinery utilized by membraneless and membrane-bound cargos are likely different. In collaboration with Jennifer Lippincott Schwartz (HHMI) and Peter Hyslop (U. Cambridge), we made a surprising discovery that RNA granules are indirectly transported long distances in axons by hitchhiking on moving lysosomes (Liao Y, Lippincott-Schwartz J* & Ward ME*, 2019, Cell (*co-corresponding authors)). To identify the potential adapters linking lysosomes to RNA granules, we used a Lamp1-APEX proximity labeling proteomic strategy in human iPSC-derived neurons. Comparing hits from our lysosome-APEX experiment against a recently-published APEX analysis of RNA granule composition revealed Annexin A11 (ANXA11), a recently discovered ALS-associated protein, as a potential adapter. In collaboration with Lucy Forrest (NINDS), structural prediction of ANXA11 revealed C-terminal membrane binding annexin domains as well as an N-terminal low-complexity (LC) domain. In vitro experiments showed that purified ANXA11 could interact with artificial lysosomes. In addition, purified ANXA11 could phase-separate in vitro, a property conferred by its N-terminal LC domain. Live cell microscopy experiments showed that ANXA11 interacted with lysosomes and RNA granules in cells, and co-trafficked in axons in cultured neurons. Purified ANXA11 was sufficient to mediate docking of purified stress granules with artificial lysosomes in vitro. In collaboration with Katie Drerup (NICHD), we showed that RNA granule/lysosome co-transport occurred in vivo in zebrafish embryos. Knockdown of ANXA11 in neurons impaired transport of RNA granules and delivery of RNA to growth cones. ALS-associated mutations in ANXA11 changed its biophysical properties (resulting in more gel-like states) and reduced its interactions with lysosomes and RNA granules. These mutations also interfered with axonal RNA granule transport and delivery of RNA to growth cones. Our observations of endolysosome-dependent axonal RNA transport is consistent with recent observations from other groups. Interestingly RNA granules hitchhike on endosomes during long-distance transport in filamentous fungi, suggesting that this mode of RNA transport is evolutionarily conserved. Together, these experiments identified a molecular relationship between lysosomes, RNA granules, and axonal transport. Our findings further suggest the possibility that dysfunctional RNA transport may be a converging mechanism of FTD/ALS, potentially disrupted by mutations in different classes of disease-associated proteins that alter lysosomal, RNA granule, or transport protein functions. Development of a CRISPRi screening platform in iPSC-derived neurons: In collaboration with Martin Kampmann at UCSF, we developed a new method to perform large-scale, forward-genetic CRISPR-inhibition (CRISPRi) screens in human iPSC-derived neurons (Ruin T, Gachechiladze M, & Ludwig C, Ward ME* & Kampmann M*, 2019, Neuron (*co-corresponding authors)). Here, we merged our highly-scalable i3Neuron differentiation technology with a potent CRISPRi screening approach developed by the Kampmann lab. Integration of a CAG-dCas9-BFP-KRAB transgene at the CLYBL safe harbor locus allowed for durable knockdown of targeted genes in both iPSCs and NGN2-differentiated neurons through lentiviral sgRNA delivery. We then performed three CRISPRi-based screens in differentiated neurons, using readouts of survival, single-cell transcriptomics, and morphology: a) A pooled survival screen in iPSCs versus neurons using a lentiviral sub-library of sgRNAs targeting 2300 kinases and druggable targets. By quantifying enrichment or depletion of sgRNAs in aged iPSCs/neurons versus the starting cell population, we identified genes for which knockdown either improved survival (protective) or reduced survival (essential). We found a shared set of genes that was essential for survival of iPSCs and neurons. Interestingly, we also discovered genes whose knockdown-related survival phenotype was cell-type specific. b) A single cell sequencing combined with CRISPRi knockdown (CROPseq) screen to interrogate how knockdowns of genes identified in our survival screen altered cellular transcriptional states. CROPseq analysis revealed cell-type specific transcriptomic responses to gene knockdowns. c) A high-content microscopy approach to characterize how gene knockdown of individual genes identified in our survival screen altered neuronal morphology and survival. These imaging studies showed that the vast majority of genes identified in our primary pooled survival screen indeed influenced cell survival, and in a number of cases also influenced neurite morphology. Finally, in a separate collaboration with Len Petrucellis lab (Mayo Clinic) we showed that CRISPRi i3Neurons could be used to reliably knockdown individual genes in neurons and assess for specific cellular phenotypes. Therefore, this system is also useful for hypothesis-directed research projects, such as monitoring accumulation of dsRNA and reduced neuronal survival in the setting of HP1alpha knockdown (Zhang YJ, Ward ME, Petrucelli L (2019) Science). We have shared these CRISPRi i3Neuron cell lines with multiple labs from the intramural, extramural, and international community. Though we continue to collaborate with the Kampmann lab, we have now built infrastructure within the IRP to independently conduct CRISPRi screens. A consortium of investigators at NCATS (Ken Cheng), NICHD (Porter, Le Pichon, Bonifacino), and NINDS (Ward) currently meet monthly as an i3Neuron workgroup to coordinate projects and refine cellular and bioinformatic protocols related to CRISPRi screens.