mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

The Neurolipid Atlas: a lipidomics resource for neurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4.

Lipid changes in the brain have been implicated in many neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's disease and Amyotrophic Lateral Sclerosis. To facilitate comparative lipidomic research across brain-diseases we established a data commons named the Neurolipid Atlas, that we have pre-populated with novel human, mouse and isogenic induced pluripotent stem cell (iPSC)-derived lipidomics data for different brain diseases. We show that iPSC-derived neurons, microglia and astrocytes display distinct lipid profiles that recapitulate in vivo lipotypes. Leveraging multiple datasets, we show that the AD risk gene ApoE4 drives cholesterol ester (CE) accumulation in human astrocytes recapitulating CE accumulation measured in the human AD brain. Multi-omic interrogation of iPSC-derived astrocytes revealed that cholesterol plays a major role in astrocyte interferon-dependent pathways such as the immunoproteasome and major histocompatibility complex (MHC) class I antigen presentation. We show that through enhanced cholesterol esterification ApoE4 suppresses immune activation of astrocytes. Our novel data commons, available at neurolipidatlas.com, provides a user-friendly tool and knowledge base for a better understanding of lipid dyshomeostasis in neurodegenerative diseases.

The disease-causing tau V337M mutation induces tau hypophosphorylation and perturbs axon morphology pathways.

Tau aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. There are disease-causing variants of the tau-encoding gene, MAPT, and the presence of tau aggregates is highly correlated with disease progression. However, the molecular mechanisms linking pathological tau to neuronal dysfunction are not well understood due to our incomplete understanding of the normal functions of tau in development and aging and how these processes change in the context of causal disease variants of tau. To address these questions in an unbiased manner, we conducted multi-omic characterization of iPSC-derived neurons harboring the MAPT V337M mutation. RNA-seq and phosphoproteomics revealed that both V337M tau and tau knockdown consistently perturbed levels of transcripts and phosphorylation of proteins related to axonogenesis or axon morphology. Surprisingly, we found that neurons with V337M tau had much lower tau phosphorylation than neurons with WT tau. We conducted functional genomics screens to uncover regulators of tau phosphorylation in neurons and found that factors involved in axonogenesis modified tau phosphorylation in both MAPT WT and MAPT V337M neurons. Intriguingly, the p38 MAPK pathway specifically modified tau phosphorylation in MAPT V337M neurons. We propose that V337M tau might perturb axon morphology pathways and tau hypophosphorylation via a "loss of function" mechanism, which could contribute to previously reported cognitive changes in preclinical MAPT gene carriers.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.

Molecular and cellular mechanisms of selective vulnerability in neurodegenerative diseases.

The selective vulnerability of specific neuronal subtypes is a hallmark of neurodegenerative diseases. In this Review, I summarize our current understanding of the brain regions and cell types that are selectively vulnerable in different neurodegenerative diseases and describe the proposed underlying cell-autonomous and non-cell-autonomous mechanisms. I highlight how recent methodological innovations - including single-cell transcriptomics, CRISPR-based screens and human cell-based models of disease - are enabling new breakthroughs in our understanding of selective vulnerability. An understanding of the molecular mechanisms that determine selective vulnerability and resilience would shed light on the key processes that drive neurodegeneration and point to potential therapeutic strategies to protect vulnerable cell populations.

Autism genes converge on microtubule biology and RNA-binding proteins during excitatory neurogenesis.

Recent studies have identified over one hundred high-confidence (hc) autism spectrum disorder (ASD) genes. Systems biological and functional analyses on smaller subsets of these genes have consistently implicated excitatory neurogenesis. However, the extent to which the broader set of hcASD genes are involved in this process has not been explored systematically nor have the biological pathways underlying this convergence been identified. Here, we leveraged CROP-Seq to repress 87 hcASD genes in a human in vitro model of cortical neurogenesis. We identified 17 hcASD genes whose repression significantly alters developmental trajectory and results in a common cellular state characterized by disruptions in proliferation, differentiation, cell cycle, microtubule biology, and RNA-binding proteins (RBPs). We also characterized over 3,000 differentially expressed genes, 286 of which had expression profiles correlated with changes in developmental trajectory. Overall, we uncovered transcriptional disruptions downstream of hcASD gene perturbations, correlated these disruptions with distinct differentiation phenotypes, and reinforced neurogenesis, microtubule biology, and RBPs as convergent points of disruption in ASD.

Toward a CRISPR understanding of gene function in human brain development.

Genome-wide association studies pinpoint genetic risk factors for neurodevelopmental disorders (NDDs), but the next challenge is to understand the mechanisms through which these genes affect brain development. Two recent CRISPR screens in human brain organoids1,2 interrogate the function of risk genes for autism spectrum disorder and other NDDs.

Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells.

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly selective ISR activators.

Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to lack of appropriate human models. Current human induced pluripotent stem cell (hiPSC)-derived neurons express very low levels of 4-repeat (4R)-tau isoforms that are normally expressed in adult brain. Here, we engineered new iPSC lines to express 4R-tau and 4R-tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes, including shared transcriptomic signatures, autophagic body accumulation, and impaired neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of Tau-seeding-induced Tau propagation, including retromer VPS29 and the UFMylation cascade as top modifiers. In AD brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade suppressed seeding-induced Tau propagation. This model provides a powerful platform to identify novel therapeutic strategies for 4R tauopathy.

Neurodegeneration enters the era of functional genomics.

There are no cures for the most common neurodegenerative diseases. None of the currently approved treatments cure or halt these conditions; rather, they address symptoms or slow disease progression. A focus on protein deposits in the brain-a hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD)-has led to the development of immunotherapy drugs. Other promising avenues of investigation include the roles of neuroinflammation in neurodegeneration. However, the clinical impact of these approaches is still uncertain. What about exploiting our knowledge of the human genome and the ability to modify it with surgically precise tools? Can functional genomics approaches in neurodegenerative disease research provide the breakthroughs we need?

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Using live cell and STORM, imaging, nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture, and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

DELE1 oligomerization promotes integrated stress response activation.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.

A hallmark of age-associated neurodegenerative diseases is the aggregation of proteins. Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to the accumulation of tau aggregates, and subsequent dysfunction and death. The mechanisms underlying cell type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways including UFMylation and GPI anchor synthesis, that control tau oligomer levels. We identify the E3 ubiquitin ligase CUL5 as a tau interactor and potent modifier of tau levels. In addition, disruption of mitochondrial function increases tau oligomer levels and promotes proteasomal misprocessing of tau. These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.

Scalable, cell type-selective, AAV-based in vivo CRISPR screening in the mouse brain.

CRISPR-based genetic screening directly in mammalian tissues in vivo is challenging due to the need for scalable, cell-type selective delivery and recovery of guide RNA libraries. We developed an in vivo adeno-associated virus-based and Cre recombinase-dependent workflow for cell type-selective CRISPR interference screening in mouse tissues. We demonstrate the power of this approach by identifying neuron-essential genes in the mouse brain using a library targeting over 2000 genes.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles.

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.

CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3ß inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-ε4, a risk variant for Alzheimer's Disease, in its effect on neuronal survival. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

Neurons require glucose uptake and glycolysis in vivo.

Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis in vivo and require glycolysis for normal function.