RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation.

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

Sonic Hedgehog receptor Patched deficiency in astrocytes enhances glucose metabolism in mice.

OBJECTIVE: Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. METHODS: We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. RESULTS: Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. CONCLUSIONS: Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

Methylglyoxal and Glyoxal as Potential Peripheral Markers for MCI Diagnosis and Their Effects on the Expression of Neurotrophic, Inflammatory and Neurodegenerative Factors in Neurons and in Neuronal Derived-Extracellular Vesicles.

Methylglyoxal (MG) and glyoxal (GO) are suggested to be associated with the development of neurodegenerative pathologies. However, their peripheral levels in relation to cognitive decline and their effects on key factors in neuronal cells are poorly investigated. The aim of this study was to determine their serum levels in MCI (mild cognitive impairment) and Alzheimer's disease (AD) patients, to analyze their effects on the neurotrophic and inflammatory factors, on neurodegenerative markers in neuronal cells and in neuronal derived-extracellular vesicles (nEVs). Our results show that MG and GO levels in serum, determined by HPLC, were higher in MCI. ROC (receiver-operating characteristic curves) analysis showed that the levels of MG in serum have higher sensitivity to differentiate MCI from controls but not from AD. Meanwhile, serum GO levels differentiate MCI from control and AD groups. Cells and nEVs levels of BDNF, PRGN, NSE, APP, MMP-9, ANGPTL-4, LCN2, PTX2, S100B, RAGE, Aß peptide, pTau T181 and alpha-synuclein were quantified by luminex assay. Treatment of neuronal cells with MG or GO reduced the cellular levels of NSE, PRGN, APP, MMP-9 and ANGPTL-4 and the nEVs levels of BDNF, PRGN and LCN2. Our findings suggest that targeting MG and GO may be a promising therapeutic strategy to prevent or delay the progression of AD.