The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible ...CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.
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•macsGESTALT is an inducible lineage recorder with efficient capture in single cells•Despite genetic competency, most cancer clones are not metastatic•Metastatic aggression peaks at specific late-hybrid EMT states•Expression of S100 genes is propagated across distinct metastatic subpopulations
Simeonov et al. develop an inducible lineage recorder, enabling simultaneous capture of lineages and transcriptomes from single cells. Lineage reconstruction in a metastatic pancreatic cancer model reveals extensive bottlenecking and subpopulation signaling, as well as specific transcriptional states associated with metastatic aggression and predictive of worse outcomes in human cancer.
Traumatic brain injury (TBI) is the strongest environmental risk factor for the accelerated development of neurodegenerative diseases. There are currently no therapeutics to address this due to lack ...of insight into mechanisms of injury progression, which are challenging to study in mammalian models. Here, we have developed and extensively characterized a head-specific approach to TBI in Drosophila, a powerful genetic system that shares many conserved genes and pathways with humans. The Drosophila TBI (dTBI) device inflicts mild, moderate, or severe brain trauma by precise compression of the head using a piezoelectric actuator. Headinjured animals display features characteristic of mammalian TBI, including severity-dependent ataxia, life span reduction, and brain degeneration. Severe dTBI is associated with cognitive decline and transient glial dysfunction, and stimulates antioxidant, proteasome, and chaperone activity. Moreover, genetic or environmental augmentation of the stress response protects from severe dTBIinduced brain degeneration and life span deficits. Together, these findings present a tunable, head-specific approach for TBI in Drosophila that recapitulates mammalian injury phenotypes and underscores the ability of the stress response to mitigate TBI-induced brain degeneration.
The aging brain is highly vulnerable to cellular stress, and neurons employ numerous mechanisms to combat neurotoxic proteins and promote healthy brain aging. The RNA modification m6A is highly ...enriched in the Drosophila brain and is critical for the acute heat stress response of the brain. Here we examine m6A in the fly brain with the chronic stresses of aging and degenerative disease. m6A levels dynamically increased with both age and disease in the brain, marking integral neuronal identity and signaling pathway transcripts that decline in level with age and disease. Unexpectedly, there is opposing impact of m6A transcripts in neurons versus glia, which conferred different outcomes on animal health span upon Mettl3 knockdown to reduce m6A: whereas Mettl3 function is normally beneficial to neurons, it is deleterious to glia. Moreover, knockdown of Mettl3 in glial tauopathy reduced tau pathology and increased animal survival. These findings provide mechanistic insight into regulation of m6A modified transcripts with age and disease, highlighting an overall beneficial function of Mettl3 in neurons in response to chronic stresses, versus a deleterious impact in glia.
m6A levels increase with age in the brain and in the brains of animals expressing human Aβ42 in neurons. Knockdown of Mettl3 in neurons decreases longevity and but increases translation efficiency by TRAP assay, and increases DNA damage. Knockdown of Mettl3 in glial cells increases longevity and decreases translation efficiency of m6A modified transcripts.
The aging brain is highly vulnerable to cellular stress, and neurons employ numerous mechanisms to combat neurotoxic proteins and promote healthy brain aging. The RNA modification m
A is highly ...enriched in the Drosophila brain and is critical for the acute heat stress response of the brain. Here we examine m
A in the fly brain with the chronic stresses of aging and degenerative disease. m
A levels dynamically increased with both age and disease in the brain, marking integral neuronal identity and signaling pathway transcripts that decline in level with age and disease. Unexpectedly, there is opposing impact of m
A transcripts in neurons versus glia, which conferred different outcomes on animal health span upon Mettl3 knockdown to reduce m
A: whereas Mettl3 function is normally beneficial to neurons, it is deleterious to glia. Moreover, knockdown of Mettl3 in glial tauopathy reduced tau pathology and increased animal survival. These findings provide mechanistic insight into regulation of m
A modified transcripts with age and disease, highlighting an overall beneficial function of Mettl3 in neurons in response to chronic stresses, versus a deleterious impact in glia.
In this issue of Neuron, Wang et al. employ an impressive multi-omics approach, including gene co-expression network analyses and drug repositioning, to normalize disease-disrupted gene modules and ...identify a therapeutically relevant driver of Alzheimer's disease.
Drosophila models have been instrumental in providing insights into molecular mechanisms of neurodegeneration, with wide application to human disease. The brain degeneration associated with traumatic ...brain injury (TBI) has been modeled in Drosophila using devices that inflict trauma on multiple parts of the fly body, including the head. However, the injuries produced by these models are not specific in location and are inconsistent between individual animals. We have recently developed a device that can be used to inflict controlled head injury to flies, resulting in physiological responses that are remarkably similar to those observed in humans with TBI. This protocol describes the construction, calibration and use of the Drosophila TBI (dTBI) device, a platform that employs a piezoelectric actuator to reproducibly deliver a force in order to briefly compress the fly head against a metal surface. The extent of head compression can be controlled through an electrical circuit, allowing the operator to set different levels of injury. The entire device can be assembled and calibrated in under a week. The device components and the necessary electrical tools are readily available and cost ~$800. The dTBI device can be used to harness the power of Drosophila genetics and perform large-scale genetic or pharmacological screens, using a 7-d post-injury survival curve to identify modifiers of injury.
Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian ...microglia. We previously reported changes in glia composition over the life of ants and fruit flies, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. Here, we show that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid‐beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity.
Ensheathing glia decrease in number during Drosophila melanogaster aging, with dysregulated expression of genes involved in lipid metabolism and apoptosis. Inhibition of apoptosis in ensheathing glia extended lifespan, improved climbing ability of aged flies, and prevented amyloid‐beta accumulation in a fly model of Alzheimer's disease.
The emergence of degenerative disease after traumatic brain injury is often described as an acceleration of normal age-related processes. Whether similar molecular processes occur after injury and in ...age is unclear. Here we identify a functionally dynamic and lasting transcriptional response in glia, mediated by the conserved transcription factor AP1. In the early post-TBI period, glial AP1 is essential for recovery, ensuring brain integrity and animal survival. In sharp contrast, chronic AP1 activation promotes human tau pathology, tissue loss, and mortality. We show a similar process activates in healthy fly brains with age. In humans, AP1 activity is detected after moderate TBI and correlates with microglial activation and tau pathology. Our data provide key molecular insight into glia, highlighting that the same molecular process drives dynamic and contradictory glia behavior in TBI, and possibly age, first acting to protect but chronically promoting disease.
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