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Despite years of intense investigation, the mechanisms underlying neuronal death in Alzheimer’s disease, remain incompletely understood. To define relevant pathways, we conducted an unbiased, genome-scale forward genetic screen for age-associated neurodegeneration in Drosophila . We also measured proteomics, phosphoproteomics, and metabolomics in Drosophila models of Alzheimer’s disease and identified Alzheimer’s genetic variants that modify gene expression in disease-vulnerable neurons in humans. We then used a network model to integrate these data with previously published Alzheimer’s disease proteomics, lipidomics and genomics. Here, we computationally predict and experimentally confirm how HNRNPA2B1 and MEPCE enhance toxicity of the tau protein, a pathological feature of Alzheimer’s disease. Furthermore, we demonstrated that the screen hits CSNK2A1 and NOTCH1 regulate DNA damage in Drosophila and human stem cell-derived neural progenitor cells. Our study identifies candidate pathways that could be targeted to ameliorate neurodegeneration in Alzheimer’s disease. In this study, Leventhal et al. integrate multi-omic data from human and Drosophila models of Alzheimer’s disease to define regulators of age-associated neurodegeneration in Alzheimer’s disease and the pathways through which they act.

Succinate dehydrogenase (SDH) supports cancer cell proliferation by enabling oxidative biosynthesis of the amino acid aspartate, yet SDH loss can also drive tumorigenesis. To cope with SDH loss, cancer cells can engage alternative aspartate synthesis pathways; however, the variables dictating pathway usage and adaptive mechanisms involved are incompletely understood. Here, we systematically profile the adaptation of SDH-knockout cancer cells and find that cells can adapt to SDH loss via at least two distinct mechanisms: suppression of respiratory complex I or upregulation of pyruvate carboxylase. Each route gives rise to distinct metabolic states with both shared and unique dependencies, but either route allows cells to overcome aspartate limitation, improve proliferative fitness, and mitigate pyrimidine-dependent replication stress. Overall, this work provides a comprehensive view of adaptive aspartate synthesis in SDH-deficient cancer cells, highlights a remarkable redox-constrained metabolic plasticity, and nominates potential metabolic vulnerabilities likely to be shared among SDH-deficient cancer cells.

Impaired availability of the amino acid aspartate can be a metabolic constraint of cell proliferation in diverse biological contexts. However, the kinetics of aspartate depletion, and its ramifications on downstream metabolism and cell proliferation, remain poorly understood. Here, we deploy the aspartate biosensor jAspSnFR3 with live cell imaging to resolve temporal relationships between aspartate and cell proliferation from genetic, pharmacological, and nutritional manipulations. In cells with impaired aspartate acquisition from mitochondrial complex I inhibition or constrained uptake in aspartate auxotrophs, we find that the proliferation defects lag changes in aspartate levels and only manifest once aspartate levels fall below a critical threshold, supporting the functional link between aspartate levels and cell proliferation in these contexts. In another context of aspartate synthesis inhibition, impairing succinate dehydrogenase (SDH), we find a more complex metabolic interaction, with initial aspartate depletion followed by a rebound of aspartate levels over time. We find that this aspartate rebound effect results from SDH inhibition disproportionately impairing pyrimidine synthesis by inhibiting aspartate transcarbamoylase (ATCase) through the dual effect of diminishing aspartate substrate availability while accumulating succinate, which functions as a competitive inhibitor of aspartate utilization. Finally, we uncover that the nucleotide imbalance from SDH inhibition causes replication stress and introduces a vulnerability to ATR kinase inhibition. Altogether, these findings identify a mechanistic role for succinate in modulating nucleotide synthesis and demonstrate how cascading metabolic interactions can unfold to impact cell function.

Genomic screens and GWAS are powerful tools for identifying disease-modifying genes, but it is often challenging to understand the pathways by which these genes function. Here, we take an integrated approach that combines network analysis and an imaging-based pooled genetic perturbation study to examine modifiers of Huntington's disease (HD). The computational analysis highlighted several genes in a subnetwork enriched for modifiers of neuronal development and morphology. To test the functional roles of these genes, we developed an experimental pipeline that allows pooled CRISPRi KD of 21 genes in human iPSC-derived neurons followed by optical analysis of genotypes, neuronal arborization, multiplexed pathway activity and morphological fingerprint readout. This approach recovered known genes involved in morphology and confirmed unexpected links from the network between several genetic modifiers of HD and morphology. Our approach overcomes challenges in pooled measurement of neuronal function and health and could be adapted for other phenotypes in HD and other neurological diseases.

Genomic screens and GWAS are powerful tools for identifying disease-modifying genes, but it is often challenging to understand the pathways by which these genes function. Here, we take an integrated approach that combines network analysis and an imaging-based pooled genetic perturbation study to examine modifiers of Huntington's disease (HD). The computational analysis highlighted several genes in a subnetwork enriched for modifiers of neuronal development and morphology. To test the functional roles of these genes, we developed an experimental pipeline that allows pooled CRISPRi KD of 21 genes in human iPSC-derived neurons followed by optical analysis of genotypes, neuronal arborization, multiplexed pathway activity and morphological fingerprint readout. This approach recovered known genes involved in morphology and confirmed unexpected links from the network between several genetic modifiers of HD and morphology. Our approach overcomes challenges in pooled measurement of neuronal function and health and could be adapted for other phenotypes in HD and other neurological diseases.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Acknowledgement updated to include NIH grants

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

Despite years of intense investigation, the mechanisms underlying neuronal death in Alzheimer's disease, the most common neurodegenerative disorder, remain incompletely understood. To define relevant pathways, we integrated the results of an unbiased, genome-scale forward genetic screen for age-associated neurodegeneration in with human and Alzheimer's disease-associated multi-omics. We measured proteomics, phosphoproteomics, and metabolomics in models of Alzheimer's disease and identified Alzheimer's disease human genetic variants that modify expression in disease-vulnerable neurons. We used a network optimization approach to integrate these data with previously published Alzheimer's disease multi-omic data. We computationally predicted and experimentally demonstrated how and enhance tau-mediated neurotoxicity. Furthermore, we demonstrated that the screen hits and regulate DNA damage in and human iPSC-derived neural progenitor cells. Our work identifies candidate pathways that could be targeted to ameliorate neurodegeneration in Alzheimer's disease.

Decreased availability of the amino acid aspartate can constrain cell function in diverse biological contexts, but the temporal interplay between aspartate, downstream metabolic changes, and functional effects remains poorly understood. Using an aspartate biosensor and live-cell imaging, we examine the interaction between aspartate abundance and cell proliferation in several models of aspartate limitation. While aspartate deficiencies intuitively interface with proliferation in some contexts, aspartate limitation from succinate dehydrogenase (SDH) inhibition causes strikingly nonintuitive dynamics resulting from an outsized impairment of pyrimidine synthesis. Mechanistically, we find that SDH loss impairs pyrimidine biosynthesis by decreasing aspartate and accumulating succinate, which competitively inhibits mammalian aspartate transcarbamylase (ATCase). This metabolic interaction persists in multiple models of SDH deficiency, causing pyrimidine insufficiency, replication stress, and sensitivity to ATR kinase inhibition. These findings define a novel role for succinate in modulating cellular nucleotide homeostasis, suggest a potential therapeutic vulnerability of SDH-deficient tumors, and demonstrate how cascading metabolic interactions can unfold to impact cell function.