A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns.

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.

Identification of RNA-RBP Interactions in Subcellular Compartments by CLIP-Seq.

Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) allows the identification of RNA targets bound by a specific RNA-binding protein (RBP) in in vivo and ex vivo experimental models with high specificity. Due to the little RNA yield obtained after cross-linking, immunoprecipitation, polyacrylamide gel electrophoresis, membrane transfer, and RNA extraction, CLIP-seq is usually performed from relatively large amounts of starting material, like cell lysates or tissue homogenates. However, RBP binding of its specific RNA targets depends on its subcellular localization, and a different set of RNAs may be bound by the same RBP within distinct subcellular sites. To uncover these RNA subsets, preparation of CLIP-seq libraries from specific subcellular compartments and comparison to CLIP-seq datasets from total lysates is necessary, yet there are currently no available protocols for this. Here we describe the adaptation of CLIP-seq to identify the specific RNA targets of an RBP (FUS) at a small subcompartment, that is, neuronal synapses, including subcompartment isolation, RBP-RNA complex enrichment, and upscaling steps.

Cytoplasmic FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and inhibitory synaptic defects.

Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization.

Synaptic FUS accumulation triggers early misregulation of synaptic RNAs in a mouse model of ALS.

Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration.

Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.

Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata.