Directly converted patient-specific induced neurons mirror the neuropathology of FUS with disrupted nuclear localization in amyotrophic lateral sclerosis

Background Mutations in the fused in sarcoma (FUS) gene have been linked to amyotrophic lateral sclerosis (ALS). ALS patients with FUS mutations exhibit neuronal cytoplasmic mislocalization of the mutant FUS protein. ALS patients fibroblasts or induced pluripotent stem cell (iPSC)-derived neurons have been developed as models for understanding ALS-associated FUS (ALS-FUS) pathology; however, pathological neuronal signatures are not sufficiently present in the fibroblasts of patients, whereas the generation of iPSC-derived neurons from ALS patients requires relatively intricate procedures. Results Here, we report the generation of disease-specific induced neurons (iNeurons) from the fibroblasts of patients who carry three different FUS mutations that were recently identified by direct sequencing and multi-gene panel analysis. The mutations are located at the C-terminal nuclear localization signal (NLS) region of the protein (p.G504Wfs*12, p.R495*, p.Q519E): two de novo mutations in sporadic ALS and one in familial ALS case. Aberrant cytoplasmic mislocalization with nuclear clearance was detected in all patient-derived iNeurons, and oxidative stress further induced the accumulation of cytoplasmic FUS in cytoplasmic granules, thereby recapitulating neuronal pathological features identified in mutant FUS (p.G504Wfs*12)-autopsied ALS patient. Importantly, such FUS pathological hallmarks of the patient with the p.Q519E mutation were only detected in patient-derived iNeurons, which contrasts to predominant FUS (p.Q519E) in the nucleus of both the transfected cells and patient-derived fibroblasts. Conclusions Thus, iNeurons may provide a more reliable model for investigating FUS mutations with disrupted NLS for understanding FUS-associated proteinopathies in ALS

The Cdc42-selective GAP Rich regulates postsynaptic development and retrograde BMP transsynaptic signaling

Inhibition of Cdc42 by dRich induces postsynaptic release of the BMP ligand Glass bottom boat

Emerging role of senescent microglia in brain aging-related neurodegenerative diseases

Abstract Brain aging is a recognized risk factor for neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), but the intricate interplay between brain aging and the pathogenesis of these conditions remains inadequately understood. Cellular senescence is considered to contribute to cellular dysfunction and inflammaging. According to the threshold theory of senescent cell accumulation, the vulnerability to neurodegenerative diseases is associated with the rates of senescent cell generation and clearance within the brain. Given the role of microglia in eliminating senescent cells, the accumulation of senescent microglia may lead to the acceleration of brain aging, contributing to inflammaging and increased vulnerability to neurodegenerative diseases. In this review, we propose the idea that the senescence of microglia, which is notably vulnerable to aging, could potentially serve as a central catalyst in the progression of neurodegenerative diseases. The senescent microglia are emerging as a promising target for mitigating neurodegenerative diseases

Loss of <i>sulf1</i>/<i>hs6st</i> causes opposite effects on transmission strength.

<p>(A) Representative excitatory junctional current (EJC) traces from control (<i>w¹¹¹⁸</i>×UH1-GAL4), <i>sulf1</i> RNAi (UH1-GAL4×UAS-CG6725) and <i>hs6st</i> RNAi (UH1-GAL4×UAS-CG4451). The nerve was stimulated (arrows) in 1.0 mM external Ca²⁺, with TEVC records (−60 mV holding potential) from muscle 6 in segment A3. Each trace averaged from 10 consecutive recordings. (B) Quantified mean EJC amplitudes (nA) for the three genotypes shown in panel A. (C) Representative traces from control (<i>w¹¹¹⁸</i>), <i>sulf1^Δ1</i> and <i>hs6st^d770</i> null alleles under the same conditions described in panel A. (D) Quantified mean EJC amplitudes (nA) for the three genotypes shown in panel C. Sample sizes are at least 11 animals per indicated genotype. Statistically significant differences calculated using student's t-test, ** p<0.01, *** p<0.001. Error bars indicate S.E.M.</p

A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling

<div><p>A <em>Drosophila</em> transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (<em>hs6st</em>) and sulfatase (<em>sulf1</em>), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of <em>hs6st</em> and <em>sulf1</em> causes opposite effects on functional synapse development, with decreased (<em>hs6st</em>) and increased (<em>sulf1</em>) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, <em>hs6st</em> and <em>sulf1</em> nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in <em>hs6st</em> and <em>sulf1</em> mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in <em>hs6st</em> and <em>sulf1</em> mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered <em>trans</em>-synaptic signaling causes functional differentiation defects.</p> </div

Loss of <i>sulf1</i> and <i>hs6st</i> causes opposite effects on WNT signaling.

<p>(A) Representative images of muscle nuclei from control (<i>w¹¹¹⁸</i>), <i>sulf1</i> and <i>hs6st</i> nulls, labeled with nuclear marker propidium iodide (PI, red) and for the C-terminus of the Wingless receptor Frizzled 2 (dFz2-C, green). Arrows indicate punctate dFz2-C nuclear labeling. Nuclei shown from muscle 6 in segment A3. (B) Quantification of the number of dFz2-C punctae per nuclei, normalized to genetic control. The total number of nuclei analyzed is indicated in each column; 119 for control (<i>w¹¹¹⁸</i>) and 163 nuclei each for <i>sulf1</i> and <i>hs6st</i> null mutants. Sample sizes are ≥9 animals per indicated genotypes. Statistically significant differences calculated using student's t-test; ** p<0.01 *** p<0.001. Error bars indicate S.E.M.</p

Glycan-related gene RNAi screen for synapse structure/function defects.

<p>Transgenic RNAi screen interrogating effects of glycan-related gene knockdown on the morphology and function of the <i>Drosophila</i> neuromuscular junction (NMJ) synapse. All VDRC UAS-RNAi lines were crossed to the UH1-GAL4 driver line. Target genes are indicated by <i>Drosophila</i> genome CG annotation number and categorized by function. Confocal imaging of co-labeled pre- and postsynaptic markers was used to quantify NMJ architecture, including branch number, bouton number and synaptic area. TEVC electrophysiology was used to quantify evoked excitatory junctional current (EJC) amplitudes. The magnitude of fold changes compared to control (<i>w¹¹¹⁸</i>×UH1-GAL4) is shown on a color scale (see legend below the two columns). Statistical significance was calculated using one-way ANOVA analysis, and displayed as p<0.05 (*), p<0.01 (**).</p

Synaptic WNT and BMP ligand abundance is modified by 6-O-S sulfation.

<p>Images show muscle 6 NMJ in segment A3 probed in non-detergent conditions, so that only extracellular protein distributions are detected. The white lines indicate cross-section planes for spatial measurements. Insets indicate single synaptic boutons at higher magnification. (A) Representative NMJ boutons from control (<i>w¹¹¹⁸</i>), <i>sulf1</i> and <i>hs6st</i> null genotypes, labeled for presynaptic anti-horseradish peroxidase (HRP, red) and anti-wingless (Wg, green). (B) Extracellular distribution of Wg across the diameter of a synaptic bouton. The Y-axis indicates intensity and the X-axis shows distance in microns. The HRP intensity profile is indicated in red; Wg intensity is shown in green. (C) Quantification of Wg mean intensity levels normalized to the HRP co-label, and to genetic control. Sample sizes are at least 15 animals per indicated genotypes. (D) Representative synaptic boutons labeled with presynaptic anti-Fasciclin II (FasII; green) and anti-Glass Bottom Boat (Gbb; red). (E) Gbb distribution across the diameter of a synaptic bouton. Y-axis indicates intensity and the X-axis shows distance in microns. FasII intensity profile is indicated in green; Gbb intensity is shown in red. (F) Quantification of Gbb mean intensity levels normalized to genetic control. Sample sizes are at least 11 independent NMJs of at least 7 animals per indicated genotypes. Statistically significant differences calculated using student's t-test and Mann-Whitney test for non-parametric data, ** p<0.01, *** p<0.001. Error bars indicate S.E.M.</p