RESUMO
A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Demência Frontotemporal/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Neurônios Motores/patologia , Proteínas do Tecido NervosoRESUMO
The microtubule-associated protein tau has a critical role in Alzheimer's disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived 'tau biosensor cells'. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.
Assuntos
Citosol/metabolismo , Exossomos/fisiologia , Lisossomos/fisiologia , Proteínas tau/metabolismo , Animais , Autofagia , Endossomos/metabolismo , Células HEK293 , Humanos , Lentivirus/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Permeabilidade , Deficiências na Proteostase , Tauopatias , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Tau is a microtubule-associated protein that is regulated by post-translational modifications. The most studied of these modifications is phosphorylation, which affects Tau's aggregation and loss- and gain-of-functions, including the interaction with microtubules, in Alzheimer's disease and primary tauopathies. However, little is known about how Tau's phosphorylation state affects its dynamics and organisation at the single-molecule level. Here, using quantitative single-molecule localisation microscopy, we examined how mimicking or abrogating phosphorylation at 14 disease-associated serine and threonine residues through mutagenesis influences the behaviour of Tau in live Neuro-2a cells. We observed that both pseudohyperphosphorylated Tau (TauE14) and phosphorylation-deficient Tau (TauA14) exhibit a heterogeneous mobility pattern near the plasma membrane. Notably, we found that the mobility of TauE14 molecules was higher than wild-type Tau molecules, while TauA14 molecules displayed lower mobility. Moreover, TauA14 was organised in a filament-like structure resembling cytoskeletal filaments, within which TauA14 exhibited spatial and kinetic heterogeneity. Our study provides a direct visualisation of how the phosphorylation state of Tau affects its spatial and temporal organisation, presumably reflecting the phosphorylation-dependent changes in the interactions between Tau and its partners. We suggest that alterations in Tau dynamics resulting from aberrant changes in phosphorylation could be a critical step in its pathological dysregulation.
Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Fosforilação , Proteínas tau/metabolismo , Imagem Individual de Molécula , Doença de Alzheimer/patologia , Microtúbulos/metabolismoRESUMO
The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer's disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer's patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.
Assuntos
Doença de Alzheimer , Exossomos , Tauopatias , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/genética , Tauopatias/metabolismo , Tauopatias/patologia , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Exossomos/genéticaRESUMO
Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.
RESUMO
Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.
Assuntos
Caulobacter crescentus , Proteínas Intrinsicamente Desordenadas , Proteínas de Bactérias/metabolismo , Condensados Biomoleculares , Caulobacter crescentus/metabolismo , Divisão Celular , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismoRESUMO
Alzheimer's disease (AD) is a proteinopathy exhibiting aggregation of ß-amyloid (Aß) as amyloid plaques and tau as neurofibrillary tangles (NFTs), whereas primary tauopathies display only a tau pathology. Aß toxicity is mediated by Fyn kinase in a tau-dependent process; however, whether Fyn controls tau pathology in diseases that lack Aß pathology remains unexplored. To address this, we generate the Tg/Fyn-/- mouse, which couples mutant tau overexpression with Fyn knockout. Surprisingly, Tg/Fyn-/- mice exhibit a near-complete ablation of NFTs, alongside reduced tau hyperphosphorylation, altered tau solubility, and diminished synaptic tau accumulation. Furthermore, Tg/Fyn-/- brain lysates elicit less tau seeding in tau biosensor cells. Lastly, the fibrillization of tau is boosted by its pseudophosphorylation at the Fyn epitope Y18. Together, this identifies Fyn as a key regulator of tau pathology independently of Aß-induced toxicity and thereby represents a potentially valuable therapeutic target for not only AD but also tauopathies more generally.