CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue.

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

Circadian autophagy drives iTRF-mediated longevity.

Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.

Kinetics of tau aggregation reveals patient-specific tau characteristics among Alzheimer's cases.

The accumulation of tau aggregates throughout the human brain is the hallmark of a number of neurodegenerative conditions classified as tauopathies. Increasing evidence shows that tau aggregation occurs in a 'prion-like' manner, in which a small amount of misfolded tau protein can induce other, naïve tau proteins to aggregate. Tau aggregates have been found to differ structurally among different tauopathies. Recently, however, we have suggested that tau oligomeric species may differ biochemically among individual patients with sporadic Alzheimer disease, and have also showed that the bioactivity of the tau species, measured using a cell-based bioassay, also varied among individuals. Here, we adopted a live-cell imaging approach to the standard cell-based bioassay to explore further whether the kinetics of aggregation also differentiated these patients. We found that aggregation can be observed to follow a consistent pattern in all cases, with a lag phase, a growth phase and a plateau phase, which each provide quantitative parameters by which we characterize the aggregation kinetics. The length of the lag phase and magnitude of the plateau phase are both dependent upon the concentration of seeding-competent tau, the relative enrichment of which differs among patients. The slope of the growth phase correlates with morphological differences in the tau aggregates, which may be reflective of underlying structural differences. This kinetic assay confirms and refines the concept of heterogeneity in the characteristics of tau proteopathic seeds among individuals with Alzheimer's disease and is a method by which future studies may characterize longitudinal changes in tau aggregation and the cellular processes which may influence these changes.

Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons.

Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau "seed" uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.

Tau molecular diversity contributes to clinical heterogeneity in Alzheimer's disease.

Alzheimer's disease (AD) causes unrelenting, progressive cognitive impairments, but its course is heterogeneous, with a broad range of rates of cognitive decline1. The spread of tau aggregates (neurofibrillary tangles) across the cerebral cortex parallels symptom severity2,3. We hypothesized that the kinetics of tau spread may vary if the properties of the propagating tau proteins vary across individuals. We carried out biochemical, biophysical, MS and both cell- and animal-based-bioactivity assays to characterize tau in 32 patients with AD. We found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble, oligomeric, seed-competent tau. Tau seeding activity correlates with the aggressiveness of the clinical disease, and some post-translational modification (PTM) sites appear to be associated with both enhanced seeding activity and worse clinical outcomes, whereas others are not. These data suggest that different individuals with 'typical' AD may have distinct biochemical features of tau. These data are consistent with the possibility that individuals with AD, much like people with cancer, may have multiple molecular drivers of an otherwise common phenotype, and emphasize the potential for personalized therapeutic approaches for slowing clinical progression of AD.

The Alzheimer Disease-Causing Presenilin-1 L435F Mutation Causes Increased Production of Soluble Aß43 Species in Patient-Derived iPSC-Neurons, Closely Mimicking Matched Patient Brain Tissue.

Familial Alzheimer disease-causing mutations in Presenilin 1 (PSEN1) are generally thought to shift the processing of APP toward longer, more amyloidogenic Aß fragments. However, certain PSEN1 mutations cause severe reduction in gamma secretase function when expressed in the homozygous state, thus challenging the amyloid hypothesis. We sought to evaluate the effects of one such mutation, PSEN1 L435F, in more physiologic conditions and genetic contexts by using human induced pluripotent stem cell (iPSC)-derived neurons from an individual with familial AD (fAD) linked to the PSEN1 L435F mutation, and compared the biochemical phenotype of the iPS-derived neurons with brain tissue obtained at autopsy from the same patient. Our results demonstrate that in the endogenous heterozygous state, the PSEN1 L435F mutation causes a large increase in soluble Aß43 but does not change the overall levels of soluble Aß40 or Aß42 when compared with control iPSC-neurons. Increased pathologically phosphorylated tau species were also observed in PSEN1-mutant iPSC-neurons. Concordant changes in Aß species were present in autopsy brain tissue from the same patient. Finally, the feasibility of using Aß43 immunohistochemistry of brain tissue to identify fAD cases was evaluated in a limited autopsy case series with the finding that strong Aß43 staining occurred only in fAD cases.

Tau impairs neural circuits, dominating amyloid-ß effects, in Alzheimer models in vivo.

The coexistence of amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the neocortex is linked to neural system failure and cognitive decline in Alzheimer's disease. However, the underlying neuronal mechanisms are unknown. By employing in vivo two-photon Ca2+ imaging of layer 2/3 cortical neurons in mice expressing human Aß and tau, we reveal a dramatic tau-dependent suppression of activity and silencing of many neurons, which dominates over Aß-dependent neuronal hyperactivity. We show that neurofibrillary tangles are neither sufficient nor required for the silencing, which instead is dependent on soluble tau. Surprisingly, although rapidly effective in tau mice, suppression of tau gene expression was much less effective in rescuing neuronal impairments in mice containing both Aß and tau. Together, our results reveal how Aß and tau synergize to impair the functional integrity of neural circuits in vivo and suggest a possible cellular explanation contributing to disappointing results from anti-Aß therapeutic trials.