ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.
Subject(s)
Amyotrophic Lateral Sclerosis , Phosphatidylinositol 3-Kinases , Animals , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons , Mutation , RNA-Binding Protein FUS/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Disease Models, AnimalABSTRACT
Cancer is a complex cellular ecosystem where malignant cells coexist and interact with immune, stromal and other cells within the tumor microenvironment (TME). Recent technological advancements in spatially resolved multiplexed imaging at single-cell resolution have led to the generation of large-scale and high-dimensional datasets from biological specimens. This underscores the necessity for automated methodologies that can effectively characterize molecular, cellular and spatial properties of TMEs for various malignancies. This study introduces SpatialCells, an open-source software package designed for region-based exploratory analysis and comprehensive characterization of TMEs using multiplexed single-cell data. The source code and tutorials are available at https://semenovlab.github.io/SpatialCells. SpatialCells efficiently streamlines the automated extraction of features from multiplexed single-cell data and can process samples containing millions of cells. Thus, SpatialCells facilitates subsequent association analyses and machine learning predictions, making it an essential tool in advancing our understanding of tumor growth, invasion and metastasis.
Subject(s)
Single-Cell Analysis , Software , Tumor Microenvironment , Single-Cell Analysis/methods , Humans , Neoplasms/pathology , Machine Learning , Computational Biology/methodsABSTRACT
Advances in stem cell science allow the production of different cell types in vitro either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between in vitro-derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.
Subject(s)
Cell Lineage/genetics , Embryo, Mammalian/cytology , Genomics , Motor Neurons/cytology , Pluripotent Stem Cells/cytology , Animals , Epigenesis, Genetic , Mice, Inbred C57BL , Motor Neurons/metabolism , Pluripotent Stem Cells/metabolism , Transcription, GeneticABSTRACT
The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.
Subject(s)
Oncogenes/physiology , Pluripotent Stem Cells/physiology , Receptors, Notch/antagonists & inhibitors , Animals , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dipeptides/pharmacology , Genes, myc , Genes, p53 , Histone-Lysine N-Methyltransferase , Humans , Keratinocytes/drug effects , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Signal Transduction/physiology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A substantial fraction of cancers evade immune detection by silencing Stimulator of Interferon Genes (STING)-Interferon (IFN) signaling. Therapeutic reactivation of this program via STING agonists, epigenetic, or DNA-damaging therapies can restore antitumor immunity in multiple preclinical models. Here we show that adaptive induction of three prime exonuclease 1 (TREX1) restrains STING-dependent nucleic acid sensing in cancer cells via its catalytic function in degrading cytosolic DNA. Cancer cell TREX1 expression is coordinately induced with STING by autocrine IFN and downstream STAT1, preventing signal amplification. TREX1 inactivation in cancer cells thus unleashes STING-IFN signaling, recruiting T and natural killer (NK) cells, sensitizing to NK cell-derived IFNγ, and cooperating with programmed cell death protein 1 blockade in multiple mouse tumor models to enhance immunogenicity. Targeting TREX1 may represent a complementary strategy to induce cytosolic DNA and amplify cancer cell STING-IFN signaling as a means to sensitize tumors to immune checkpoint blockade (ICB) and/or cell therapies. SIGNIFICANCE: STING-IFN signaling in cancer cells promotes tumor cell immunogenicity. Inactivation of the DNA exonuclease TREX1, which is adaptively upregulated to limit pathway activation in cancer cells, recruits immune effector cells and primes NK cell-mediated killing. Targeting TREX1 has substantial therapeutic potential to amplify cancer cell immunogenicity and overcome ICB resistance. This article is featured in Selected Articles from This Issue, p. 695.
Subject(s)
Exodeoxyribonucleases , Membrane Proteins , Phosphoproteins , Signal Transduction , Exodeoxyribonucleases/genetics , Mice , Phosphoproteins/metabolism , Phosphoproteins/genetics , Humans , Animals , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neoplasms/immunology , Neoplasms/genetics , Neoplasms/drug therapy , Interferons/metabolism , Cell Line, Tumor , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolismABSTRACT
Background: Cancer is a complex cellular ecosystem where malignant cells coexist and interact with immune, stromal, and other cells within the tumor microenvironment. Recent technological advancements in spatially resolved multiplexed imaging at single-cell resolution have led to the generation of large-scale and high-dimensional datasets from biological specimens. This underscores the necessity for automated methodologies that can effectively characterize the molecular, cellular, and spatial properties of tumor microenvironments for various malignancies. Results: This study introduces SpatialCells, an open-source software package designed for region-based exploratory analysis and comprehensive characterization of tumor microenvironments using multiplexed single-cell data. Conclusions: SpatialCells efficiently streamlines the automated extraction of features from multiplexed single-cell data and can process samples containing millions of cells. Thus, SpatialCells facilitates subsequent association analyses and machine learning predictions, making it an essential tool in advancing our understanding of tumor growth, invasion, and metastasis.
ABSTRACT
Genetic mutations that cause adult-onset neurodegenerative diseases are often expressed during embryonic stages, but it is unclear whether they alter neurodevelopment and how this might influence disease onset. Here, we show that the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), a repeat expansion in C9ORF72, restricts neural stem cell proliferation and reduces cortical and thalamic size in utero. Surprisingly, a repeat expansion-derived dipeptide repeat protein (DPR) not known to reduce neuronal viability plays a key role in impairing neurodevelopment. Pharmacologically mimicking the effects of the repeat expansion on neurodevelopment increases susceptibility of C9ORF72 mice to motor defects. Thus, the C9ORF72 repeat expansion stunts development of the brain regions prominently affected in C9ORF72 FTD/ALS patients.
Subject(s)
Amyotrophic Lateral Sclerosis , C9orf72 Protein , Frontotemporal Dementia , Animals , Mice , Amyotrophic Lateral Sclerosis/genetics , C9orf72 Protein/genetics , Dipeptides , Frontotemporal Dementia/genetics , MutationABSTRACT
Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.
Subject(s)
Fibroblasts/physiology , Motor Neurons/physiology , Transcription, Genetic/physiology , Animals , Cell Proliferation , Cellular Reprogramming , DNA Topoisomerases/metabolism , Epigenesis, Genetic , Gene Expression Regulation , HEK293 Cells , Humans , Induced Pluripotent Stem Cells , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, BiologicalABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects - impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.
Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Autophagosomes/drug effects , Motor Neurons/drug effects , Protein C/administration & dosage , Adult , Aged , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/pathology , Animals , Autophagosomes/immunology , Autophagy/genetics , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , CHO Cells , Cells, Cultured , Cricetulus , Disease Models, Animal , Female , Gain of Function Mutation , Humans , Induced Pluripotent Stem Cells , Loss of Function Mutation , Lymphocytes , Male , Mice , Middle Aged , Motor Neurons/immunology , Motor Neurons/pathology , Primary Cell Culture , Protein C/genetics , Proteostasis/drug effects , Proteostasis/immunology , Receptor, PAR-1/agonists , Receptor, PAR-1/metabolism , Receptors, Glutamate/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/geneticsABSTRACT
Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.
Subject(s)
C9orf72 Protein/genetics , Neurodegenerative Diseases/genetics , Ribosomal Proteins/genetics , Animals , DNA Repeat Expansion/genetics , Humans , Protein BiosynthesisABSTRACT
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.
Subject(s)
C9orf72 Protein/genetics , Active Transport, Cell Nucleus/genetics , Amyotrophic Lateral Sclerosis/etiology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , C9orf72 Protein/metabolism , C9orf72 Protein/toxicity , CRISPR-Cas Systems , DNA Repeat Expansion , Endoplasmic Reticulum Stress/genetics , Frontotemporal Dementia/etiology , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , Gene Knockout Techniques , HeLa Cells , Humans , K562 Cells , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Microsatellite Repeats , Motor Neurons/metabolism , Thioredoxins/genetics , Thioredoxins/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , C9orf72 Protein/genetics , Frontotemporal Dementia/genetics , Nerve Degeneration/genetics , rab5 GTP-Binding Proteins/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , DNA Repeat Expansion/genetics , Disease Models, Animal , Endosomes/genetics , Frontotemporal Dementia/pathology , Gene Expression Regulation/genetics , Haploinsufficiency/genetics , Humans , Introns/genetics , Motor Neurons/metabolism , Motor Neurons/pathology , Mutation , Nerve Degeneration/physiopathologyABSTRACT
Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.