Highlights d RNA granules ''hitchhike'' on motile lysosomes during longdistance transport d ANXA11 binds to RNA and lysosomes via phase separating and membrane binding domains d ANXA11 tethers RNA granules to lysosomes and is required for axonal RNA transport d ALS-associated ANXA11 mutations impair its tethering function and RNA transport
Highlights d A CRISPR interference platform for genetic screens in human iPSC-derived neurons d Survival screens uncover genes essential for neurons, but not iPSCs or cancer cells d Single-cell RNA-seq screens reveal distinct neuronal roles for ubiquitous genes d Arrayed high-content screens uncover genes controlling neuronal morphology
INTRODUCTION: Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that share clinical and neuropathological features. Furthermore, the most common genetic cause of both FTD and ALS is a GGGGCC (G4C2) repeat expansion in the C9orf72 gene. This repeat expansion leads to several abnormalities, including C9orf72 haploinsufficiency, the accumulation of repeat RNA, and the production of five aggregation-prone proteins composed of repeating dipeptides. However, the contribution of these abnormalities to disease pathogenesis remains unresolved. RATIONALE: Among the five dipeptide repeat proteins nonconventionally translated from expanded G4C2 repeats, proline-arginine (PR) repeat proteins [poly(PR) proteins] have proven especially toxic in various model systems. Their involvement in C9orf72-associated FTD and ALS (c9FTD/ALS) has nevertheless been questioned because poly(PR) pathology is relatively infrequent in c9FTD/ALS patient brains. Postmortem tissues, however, represent end-stage disease and do not necessarily reflect early events in the disease process. Therefore, we generated mice that express poly(PR) in the brain to evaluate the temporal consequences of its expression in a mammalian in vivo model. More specifically, we engineered mice to express green fluorescent protein (GFP)–conjugated (PR)50 (a 50-repeat PR protein) or GFP via intracerebroventricular administration of adeno-associated viral vectors and then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice. RESULTS: We found that ~60% of poly(PR)- expressing mice died by 4 weeks of age and had significantly decreased brain and body weights at death compared with age-matched GFP control mice. Poly(PR) mice that escaped premature death developedmotor andmemory impairments, likely as a consequence of their progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis. In investigating the mechanisms by which poly(PR) caused neurodegeneration and functional deficits, we found that poly(PR) localized to heterochromatin (highly condensed regions of transcriptionally silent chromatin) and caused abnormal histone H3 methylation, features that we also detected in brain tissues from patients with c9FTD/ALS. Additionally, we observed aberrations in nuclear lamins and heterochromatin protein 1α (HP1α), key proteins thatmaintain heterochromatin structure and regulate gene silencing. Nuclear lamina invaginations and decreased HP1a protein expression were seen in poly(PR)-positive cells in poly(PR) mice, and in vitro studies demonstrated that poly(PR) disrupted HP1α liquid phases. Because poly(PR)-induced histone H3 posttranslational modifications, lamin invaginations, and decreased HP1α levels could profoundly affect gene expression, we compared transcriptome profiles between control and poly(PR) mice. As well as analyzing differentially expressed genes, we examined repetitive element expression given that repetitive...
CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSCderived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.2 4 systematic dissection of normal and disease states of neurons, and highlight the potential of interrogating human cell biology and gene function in iPSC-derived differentiated cell types. RESULTS Robust CRISPR interference in human iPSC-derived neuronsAs a first step towards a high-throughput screening platform in neurons, we developed a scalable CRISPRi-based strategy for robust knockdown of endogenous genes in homogeneous populations of human iPSC-derived neurons. We built on our previously described i 3 Neuron (i 3 N) platform, which enables large-scale production of iPSC-derived glutamatergic neurons. Central to this platform is an iPSC line with an inducible Neurogenin 2 (Ngn2) expression cassette in the AAVS1 safe-harbor locus (Fernandopulle et al., 2018;Wang et al., 2017). To enable stable CRISPRi in iPSC-derived neurons, we generated a plasmid (pC13N-dCas9-BFP-KRAB) to insert an expression cassette for CAG promoter-driven dCas9-BFP-KRAB into the CLYBL safe harbor locus, which enables robust transgene expression throughout neuronal differentiation at higher levels than the AAVS1 locus (Cerbini et al., 2015) ( Fig. 1A). We then integrated this cassette into our i 3 N iPSC line, and called the resulting monoclonal line CRISPRi-i 3 N iPSCs. A normal karyotype was confirmed for CRISPRi-i 3 N iPSCs ( Fig. S1A).To validate CRISPRi activity, we transduced these iPSCs with a lentiviral construct expressing an sgRNA targeting the transferrin receptor gene (TFRC). Knockdown of TFRC mRNA was robust in iPSCs and in i 3 Neurons for several weeks after differentiation (Fig. 1B,C). We also validated knockdown of three additional genes, UBQLN2 (Fig. 1D,E), GRN (Fig. 1F,G) and CDH2 (Fig. S1B) by qRT-PCR, Western blot, and/or immunofluorescence. Our platform thus enables potent CRISPRi knockdown of endogenous genes in iPSC-derived neurons.Since CRISPRn-associated DNA damage has been found to be highly toxic to iPSCs (Ihry ...
Apolipoprotein E (apoE) is associated with lipoproteins in the cerebrospinal fluid (CSF). APOE4 increases and APOE2 decreases the risk for Alzheimer disease (AD) compared to the risk associated with APOE3 Because apoE4 is less efficient at cholesterol efflux than apoE2 or apoE3 in vitro, we hypothesized that APOE genotype may affect apoE particle size in vivo and that these size differences may be related to AD risk. We used nondenaturing gel electrophoresis to test for differences in the size of apoE complexes in human CSF samples of various APOE genotypes and created profiles of each sample to compare the patterns of apoE distribution. For middle-aged adults with no dementia, APOE 2.3 individuals had significantly larger apoE complexes than APOE 3.3 subjects, who had significantly larger apoE complexes than APOE 3.4 and APOE 4.4 individuals. Similarly, in an independent cohort of older adults, CSF apoE complexes of APOE4-positive individuals were smaller than those of the APOE4-negative individuals. Compared to individuals with no dementia, those with the mildest stages of dementia had similar sized CSF apoE complexes. These results identify a novel phenotypic difference in the size of CSF apoE complexes in middle age that correlate with the risk of AD later in life.
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