M6A-dependent RNA condensation underlies FUS autoregulation and can be harnessed for ALS therapy development

Mutations in the FUS gene cause aggressive amyotrophic lateral sclerosis (ALS-FUS). Beyond mRNA, FUS generates partially processed transcripts retaining introns 6 and 7. We demonstrate that these FUSint6&7-RNA molecules form nuclear condensates, scaffolded by the highly structured intron 7 and associated with nuclear speckles. Using hybridization-proximity labeling proteomics, we show that the FUSint6&7-RNA condensates are enriched for splicing factors and the N6-methyladenosine (m6A) reader YTHDC1. These ribonucleoprotein structures facilitate posttranscriptional FUS splicing and depend on m6A/YTHDC1 for integrity. In cells expressing mutant FUS, FUSint6&7-RNAs become hypermethylated, which in turn stimulates their condensation and splicing. We further show that FUS protein is repelled by m6A. Thus, ALS-FUS mutations may cause abnormal activation of FUS posttranscriptional splicing through altered RNA methylation. Notably, ectopic expression of FUS intron 7 sequences dissolves endogenous FUSint6&7-RNA condensates, down-regulating FUS mRNA and protein. Our findings reveal a condensation-dependent mechanism regulating FUS splicing, with possible therapeutic implications for ALS

Chronically stressed or stress-preconditioned neurons fail to maintain stress granule assembly

Dysregulation of stress granules (SGs) and their resident proteins contributes to pathogenesis of a number of (neuro)degenerative diseases. Phosphorylation of eIF2α is an event integrating different types of cellular stress and it is required for SG assembly. Phosphorylated eIF2α (p-eIF2α) is upregulated in the nervous system in some neurodegenerative conditions. We found that increasing p-eIF2α level by proteasomal inhibition in cultured cells, including mouse and human neurons, prior to a SG-inducing stress (‘stress preconditioning’), limits their ability to maintain SG assembly. This is due to upregulation of PP1 phosphatase regulatory subunits GADD34 and/or CReP in preconditioned cells and early decline of p-eIF2α levels during subsequent acute stress. In two model systems with constitutively upregulated p-eIF2α, mouse embryonic fibroblasts lacking CReP and brain neurons of tau transgenic mice, SG formation was also impaired. Thus neurons enduring chronic stress or primed by a transient mild stress fail to maintain p-eIF2α levels following subsequent acute stress, which would compromise protective function of SGs. Our findings provide experimental evidence on possible loss of function for SGs in certain neurodegenerative diseases

Molecular pathomechanisms of Amyotrophic Lateral Sclerosis caused by FUS mutations

Amyotrophic lateral sclerosis (ALS) is the most common type of motor neuron disease affecting both upper and lower motor neurons. About 10% of ALS cases run in families with known genetic background, and mutations in the Fused in Sarcoma (FUS) gene are responsible for about 5% of the fALS cases (ALS-FUS). Despite normal FUS is a predominantly nuclear protein, mutant FUS is found to accumulate and aggregate in the cytoplasm of affected neurons and glial cells in ALS-FUS. It is generally believed that mutations are the primary cause of FUS protein mislocalisation, and additional stresses are required to trigger the formation of insoluble FUS aggregates (FUSopathy). However, no clear consensus has been achieved on many important questions. For instance, what are the consequences of FUS protein mutation for its nuclear function and how do they contribute to ALS-FUS development? What is the nature of the stress that promotes the massive protein accumulation and inclusion formation in the cytoplasm? This thesis attempts to address these questions using novel cellular models with targeted modifications of the FUS gene. It is demonstrated that the presence of endogenous mutant FUS protein in the nucleus causes hyper-assembly of structurally and functionally abnormal paraspeckles - nuclear bodies assembled on the long non-coding RNA called Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysfunctional paraspeckles together with accumulation of NEAT1 outside paraspeckles might contribute to the disease severity. Stresses capable of triggering cytoplasmic FUS aggregates are also investigated, and as a result, antiviral immune response has emerged as a potent stress promoting formation of persistent cytoplasmic FUS-positive assemblies. In addition, type I interferon expressed during antiviral response is found to cause FUS protein accumulation by increasing FUS mRNA stability. I propose a multi-step model where antiviral immune response serves as the "second hit" provoking FUSopathy. This thesis offers novel insights into the cellular and molecular events leading to the initiation and progression of ALS-FUS, which should help inform the development of therapeutic strategies in the future

P53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation

Glioblastoma (GBM) is a highly lethal brain tumor presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as high-grade disease that typically harbors EGFR, Pten and Ink4a/Arf mutations, and the secondary GBM subtype evolves from the slow progression of low-grade disease that classically possesses PDGF and p53 events1–3. Here, we show that concomitant CNS-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with striking clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted p53 and Pten mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of p53 as well the expected Pten mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives elevated c-Myc levels and its associated signature. Functional studies validated increased c-Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of p53-Pten null NSCs as well as tumor neurospheres (TNSs) derived from this model. c-Myc also serves to maintain robust tumorigenic potential of p53-Pten null TNSs. These murine modeling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumor suppressor mutation profile in human primary GBM and establish c-Myc as a key target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential

Investigation on data fusion of sun-induced chlorophyll fluorescence and reflectance for photosynthetic capacity of rice

Studying crop photosynthesis is crucial for improving yield, but current methods are labor-intensive. This research aims to enhance accuracy by combining leaf reflectance and sun-induced chlorophyll fluorescence (SIF) signals to estimate key photosynthetic traits in rice. The study analyzes 149 leaf samples from two rice cultivars, considering reflectance, SIF, chlorophyll, carotenoids, and CO2 response curves. After noise removal, SIF and reflectance spectra are used for data fusion at different levels (raw, feature, and decision). Competitive adaptive reweighted sampling (CARS) extracts features, and partial least squares regression (PLSR) builds regression models. Results indicate that using either reflectance or SIF alone provides modest estimations for photosynthetic traits. However, combining these data sources through measurement-level data fusion significantly improves accuracy, with mid-level and decision-level fusion also showing positive outcomes. In particular, decision-level fusion enhances predictive capabilities, suggesting the potential for efficient crop phenotyping. Overall, sun-induced chlorophyll fluorescence spectra effectively predict rice's photosynthetic capacity, and data fusion methods contribute to increased accuracy, paving the way for high-throughput crop phenotyping